| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biochemistry and Biophysics |
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 Canada [M. R., H. J. A., L. L., X. J., C. M. B.]; Departments of Chemistry and Oceanography (EOS), University of British Columbia, Vancouver, V6T 1Z1 Canada [B. C., L. X., R. J. A.]; and SeaTek Marine Biotechnology, Inc., Surrey, British Columbia, V4A 7M4 Canada [M. T. K.]
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
- and ß-tubulin into microtubules. At
high concentrations, the Vinca alkaloids and most other
antimitotics cause complete microtubule depolymerization, whereas the
taxanes cause bundling of microtubules by stabilizing them against
depolymerization. At low concentrations, neither depolymerization nor
bundling is observed, but there is sufficient alteration in the
dynamics of tubulin loss or addition at the ends of mitotic spindle
microtubules to prevent the spindle from carrying out its function of
attaching to and segregating the chromosomes, and cells arrest in
mitosis (3
, 4)
. Prolonged arrest eventually leads to cell
death, either in mitosis or after an eventual escape from mitotic
arrest (5
, 6) . Another class of antimitotic agents,
represented by estramustine, does bind tubulin (7)
but may
also bind microtubule-associated proteins and prevent them from
regulating interactions between tubulin polymers (8)
.
Agents that are not known to interact with microtubules, such as
inhibitors of protein phosphatases 1 and 2A and mitotic kinesin
inhibitors, can also arrest cells in mitosis (9, 10, 11)
. The Vinca alkaloids were isolated from the periwinkle plant, which originally attracted attention because of reported hypoglycemic properties. However, periwinkle extracts showed no antidiabetic action but were found to prolong the life of mice bearing a transplantable lymphocytic leukemia (1) . This led to the identification of vincristine and vinblastine. Paclitaxel was isolated from the bark of the Pacific yew tree, an extract of which showed antineoplastic activity in the NCI3 large-scale screen (2) . Vinorelbine and docetaxel are semisynthetic analogues.
These drugs, although extremely valuable, are not ideal. They have
numerous toxicities, principally myelosuppression and neurotoxicity.
More importantly, many cancers are inherently resistant to these drugs
or become so during prolonged treatment (1
, 2)
. This is
often the result of multidrug resistance caused by overexpression of
P-glycoprotein, which functions as a drug efflux pump. Other sources of
resistance include increased expression of tubulin isotypes to which a
particular drug binds less effectively and alterations in - and
ß-tubulin structure, by mutation or posttranslational
modification, that reduce binding.
Antimitotics with different chemical structures might show increased specificity to mitotic microtubules rather than neuronal microtubules and reduce unwanted side effects and might be effective against resistant cancers. Many other antimitotics have been discovered, some of which show promise in preclinical studies or have entered clinical trials (12) . However, they were discovered either by serendipity or by cytotoxicity screening, or because they showed patterns of cytotoxic activity against panels of cancer cell lines similar to patterns shown by other antimitotic agents (13) . The search for better antimitotics would be greatly aided by rational assays for use in drug screens.
We have developed a rapid and reliable cell-based screen for antimitotic agents. In this report, we describe the assay, its application to a screen of >24,000 natural extracts, and the purification and characterization of paclitaxel analogues and new rhizoxin and eleutherobin analogues.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
ELISA of Mitotic Cells.
After incubation with extracts, the cell culture medium was withdrawn
carefully using a pipettor. This did not result in any loss of
the rounded-up mitotic cells, which remained attached to the plates.
The cells were lysed by adding 100 µl of ice-cold lysis buffer (1
mM EGTA, pH 7.4, 0.5 mM phenylmethylsulfonyl
fluoride) and by pipetting up-and-down 10 times. The cell lysates were
transferred to 96-well PolySorp plates (Nunc) and dried completely in a
stream of air at about 37°C from a hair dryer. Vacant protein binding
sites were blocked by adding 200 µl/well of antibody buffer [10
mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1
mM phenylmethylsulfonyl fluoride, and 3% (w/v) dried
nonfat milk (Carnation)] for 1 h at room temperature. This was
removed and replaced with 100 µl of antibody buffer containing
0.1–0.15 µg/ml TG-3 monoclonal antibody (15
, 16)
. This
antibody recognizes a phosphoepitope on nucleolin that is present only
at mitosis and was provided by Dr. Peter Davies (Albert Einstein
College of Medicine, Bronx, NY). After 16–20 h incubation at 4°C,
the antibody solution was removed, and the wells were rinsed twice with
200 µl of 10 mM Tris-HCl (pH 7.4), 0.02% Tween 20.
HRP-conjugated goat antimouse IgM secondary antibody (Southern
Biotechnology Associates) was added at a dilution of 1:500. After
overnight incubation at 4°C, the antibody solution was removed, and
the wells were rinsed three times with 200 µl of 10 mM
Tris-HCl (pH 7.4), 0.02% Tween 20. One hundred µl of 120
mM Na2HPO4, 100
mM citric acid (pH 4.0) containing 0.5 µg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.01%
hydrogen peroxide was added for 1 h at room temperature, and
absorbance at 405 nm was determined using a Dynex MRX plate reader.
ELICA of Mitotic Cells.
After incubation with extracts, the medium was withdrawn carefully
using a pipettor, and 100 µl of 10 mM Tris-HCl (pH 7.4),
150 mM NaCl, containing 3.7% formaldehyde, were added to
fix the cells for 30 min at 4°C. The fixative was removed and
replaced with 100 µl of cold (-20°C) methanol for 5 min to
permeabilize the fixed cells. The methanol was removed, and the wells
were rinsed briefly with 200 µl of antibody buffer. Then, 100 µl of
antibody buffer containing 0.1–0.15 µg/ml TG-3 monoclonal antibody
and HRP-conjugated goat antimouse IgM secondary antibody at a dilution
of 1:500 was added for 16–20 h at 4°C. The plates were washed twice
with 200 µl of 10 mM Tris-HCl (pH 7.4), 0.02% Tween 20.
One hundred µl of 120 mM
Na2HPO4, 100 mM
citric acid (pH 4.0) containing 0.5 µg/ml
2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.01%
hydrogen peroxide were added for 1 h at room temperature, and
absorbance at 405 nm was measured. Additional information about this
assay is provided in "Results."
Sample Collection and Extract Preparation.
Approximately 250 g each of marine invertebrates were collected by
hand, using scuba, from the cold temperate waters of the Pacific Ocean
along the coast of British Columbia, from tropical Pacific Ocean reefs
off Motupore and Madang in Papua New Guinea, and from tropical waters
off the island of Dominica in the Caribbean. Samples were deep frozen
on site and transported to Vancouver over dry ice. Voucher samples of
each invertebrate are stored in methanol at -20°C at the University
of British Columbia for taxonomic identification. Marine microorganisms
were isolated from the invertebrates on site using various marine
culture media, and pure cultures were grown as lawns on solid agar
marine media in 10-cm Petri plates for several days and then
freeze-dried.
Extracts of invertebrates were prepared by homogenizing in methanol
200 g of each sample. The homogenates were filtered and concentrated
to dryness in vacuo to give a gummy residue. Extracts of
microorganisms were prepared by extracting the freeze-dried culture
(cells and agar) multiple times with dry methanol:acetone, followed by
lyophilization. A small amount of each extract was dissolved in DMSO
for the antimitotic screen. Extracts of terrestrial plants were
obtained from the Open Repository Program of the Natural Products
Repository of the NCI Developmental Therapeutics Program as 500-µg
samples that were dissolved in 100 µl of DMSO. All diluted extracts
were stored at -20°C.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
. This protein has been identified as a mitotically phosphorylated form
of nucleolin (17)
. Densitometric scanning of the bands
showed a 27-fold difference in intensity between nocodazole-treated and
untreated cells, corresponding well to the difference in the number of
mitotic cells in the two samples: 80% for the nocodazole-treated
sample and 3% for the untreated sample, as measured by microscopy.
|
We first tested the suitability of the ELISA for quantifying the
activity of antimitotic agents. MCF-7 cells were incubated for 20 h with different concentrations of the antimitotic drug paclitaxel, and
the proportion of cells arrested in mitosis was measured by counting
mitotic figures in the microscope and by ELISA. Paclitaxel induced
mitotic arrest in a concentration-dependent manner with half-maximal
activity at 10 nM measured by microscopy (Fig. 2A)
and at 4 nM measured by ELISA (Fig. 2B)
.
|
The ELICA was tested as above. Dose-dependent arrest of cells in
mitosis by paclitaxel was detected by ELICA with half-maximal activity
at 1.5 nM (Fig. 2)
. The ELICA showed a higher signal at low
paclitaxel concentrations and a lower signal at high concentrations
than did the ELISA (Fig. 2B)
. These differences probably
resulted from higher nonspecific staining of interphase cells because
of reduced washing and from lower specific staining of mitotic cells
because of fixation and reduced antibody incubation times.
Nevertheless, the ELICA consistently showed sufficient difference in
absorbance between cells treated or not with antimitotic agents to
allow unambiguous detection of mitotic cells. Measurements obtained by
ELICA consistently showed smaller SDs than obtained by ELISA, probably
because the reduced number of manipulations reduced experimental
variation.
Screening of Natural Extracts.
We first tested the suitability of the ELISA for drug screening using a
small selection of crude extracts from marine microorganisms (Table 1)
. Of the 264 extracts tested, 261 showed no activity, giving absorbance
readings not statistically different from those of untreated cells.
Three extracts clearly showed activity, with absorbance readings of
1.135, 1.437, and 1.245, close to the values obtained with nocodazole
as a positive control.
|
Finally, we screened crude extracts of terrestrial plants from the NCI
Natural Products Repository by ELICA. The suitability of the ELICA for
drug screening is illustrated in Table 2
, which displays a screen of 264 plant extracts from three randomly
selected 96-well plates. Five extracts showed activity, with absorbance
readings close to or higher than those obtained with nocodazole. These
positive readings were well above those obtained with negative controls
or extracts showing no activity. Of 21,600 plant extracts tested in
this manner, 100 showed activity, all of which were confirmed to be
positive by microscopy.
|
Identification of New Rhizoxin Analogues.
Marine bacterial isolate MK7020 collected off the coast of British
Columbia was identified as a Pseudomonas sp. by gas
chromatographic analysis of cellular fatty acids. The active compounds
1 and 2 (Fig. 3)
were purified by chromatographic procedures using the ELISA to guide
fractionation. The two other microbial extracts were found to be
independent isolates of the same Pseudomonas species and
contained the same active compounds as MK7020.
|
. Compound 1 showed half-maximal antimitotic activity
(IC50) at 52 nM, as determined by
ELISA (data not shown). Compound 2 is a new 
-lactone
seco-hydroxy acid and had an IC50 of 8
nM (data not shown).
Identification of New Eleutherobin Analogues.
An extract of octocoral Erythropodium cf. caribaeorum
collected from shallow reefs near Dominica showed antimitotic activity
and bundling of microtubules. The active compounds 3–10
(Fig. 4)
were isolated, and their chemical structure was elucidated as
described in detail elsewhere (22)
.
|
The antimitotic activity profile of these compounds determined by ELICA
is shown in Fig. 5
. Eleutherobin (3) had an IC50 of 100
nM. The activity of Z-eleutherobin
(7) was close, with an IC50 of 250
nM. Desmethyleleutherobin (8) and
isoeleutherobin A (6) were slightly more potent than
eleutherobin, with IC50 of 20 and 50
nM, respectively. Desacetyleleutherobin
(5) was slightly less potent, with an
IC50 of 400 nM.
Sarcodictyin A (4) showed lower activity, with an
IC50 of 2 µM.
Caribaeoside (9) and caribaeolin
(10) were considerably less potent, with an
IC50 of 20 µM for both
compounds.
|
by analysis of their nuclear magnetic resonance data and comparison
with published values (26
, 27)
. Compounds 11
(IC50, 0.3 µM) and
12 (IC50, 10
µM) were much less potent than paclitaxel
(IC50, 1.5 nM).
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
The ELISA and the ELICA procedures both allow unambiguous detection of antimitotic activity in crude natural extracts. The ELICA was used for most of the screening described here because it is faster, less labor-intensive, and less costly than the ELISA.
Our screen of over 24,000 crude extracts from different natural sources identified unambiguously 119 with antimitotic activity. The absence of false-positive results was confirmed by microscopy, and all five positive crude extracts that were subjected to further study yielded known or novel antimitotic agents; three extracts from the pilot screen contained members of the rhizoxin family, one marine invertebrate extract contained compounds related to eleutherobin, and a tree extract contained paclitaxel analogues.
Structure-Antimitotic Activity Relationships.
The assay is useful not only for identifying and purifying antimitotics
but also for providing a quantitative measure of their antimitotic
activity. This is a helpful indicator of a compound’s pharmacological
potential because it measures not simply the interaction of the
compound with its target, as an in vitro assay would do, but
its ability to interact with its target within a cell. We used it to
compare the antimitotic activity of different analogues of rhizoxin,
eleutherobin, and paclitaxel.
Rhizoxin is a 16-membered macrolide isolated in 1984 (29)
and later found to cause the accumulation of cells in mitosis
(30
, 31) and to inhibit microtubule assembly (31
, 32)
. Rhizoxin is very cytotoxic to cancer cells in
vitro or in mice (20
, 30) , including cell lines
resistant to the Vinca alkaloids (30)
. It has
been the subject of several Phase I and II clinical trials, but results
have been disappointing (reviewed in Ref. 33
). To the best
of our knowledge, the seco-hydroxy acid 2 was not known
previously as a natural product, having been reported in the patent
literature only as a semisynthetic derivative of the corresponding
-lactone. WF-1360C (1) was 15-fold less toxic to P388
cells than rhizoxin (20)
. It differs from rhizoxin by the
presence of a hydroxyl group instead of a methoxyl at C-17 and the
absence of the two epoxides at C-2 to C-3 and C-11 to C-12. Compound
2 retains the methoxyl and one epoxide but has an open
lactone ring. Comparison of the antimitotic activity of WF-1360C
(IC50, 52 nM) to that of
compound 2 (IC50, 8
nM) and to published cytotoxicity data for other
analogues (20
, 32) indicates that a closed lactone
ring is not required for antimitotic activity and that the presence of
a methoxyl substituent at C-17 contributes to the high potency of
rhizoxin.
Eleutherobin was identified as a compound with paclitaxel-like properties in 1997 (23) , but sarcodictyins A-D were the first members of the eleutherobin class of compounds to be identified (25 , 34) , their paclitaxel-like properties were recognized only later (35) . Sarcodictyin A (4) was 20-fold less active than eleutherobin (3), indicating that the C-15 ß-linked 2'-O-acetyl-D-arabinopyranose side chain or the C-4 methoxyl group is important for antimitotic activity. Desmethyleleutherobin (8) was active, showing that it is the C-15 side chain and not the C-4 methoxyl that is required. Desacetyleleutherobin (5) and isoeleutherobin A (6) showed activity similar to eleutherobin, indicating that the acetyl group does not contribute importantly to activity. Therefore, although the sugar moiety is not absolutely required for antimitotic activity, it contributes to the high potency of eleutherobin.
Isomerization of the C-2' to C-3' double bond of the C-8 side chain of Z-eleutherobin (7) had little effect on the antimitotic activity of the compound, showing that the E configuration in eleutherobin is not required for antimitotic activity. Desmethyleleutherobin (8) was the most active of the compounds tested, suggesting that the C-4 hydroxyl might enhance activity through additional hydrogen bonding, or that the C-4 methoxyl somehow hinders the activity of eleutherobin. Caribaeoside (9) was 200-fold less active than eleutherobin, revealing the importance of the C-11 to C-13 segment for antimitotic activity. Caribaeolin (10) differs from caribaeoside (9) only in the C-3 side chain, and the activities of these compounds are similar. Likewise, sarcodictyin A differs from desmethyleleutherobin only in the C-3 side chain, but its activity is lower than that of desmethyleleutherobin. These data indicate that the C-15 acetyl-D-arabinopyranose can be replaced with an acetoxy functionality without significant loss of activity, confirming earlier data with synthetic analogues (36 , 37) , but not with a methyl ester.
Thirteen synthetic eleutherobin analogues have recently been described and tested in tubulin polymerization and cytotoxicity assays (36, 37, 38) . Overall, these studies underlined the importance of the C-8 and C-3 side chains for activity, the C-8 side chain being essential and the sugar or another bulky substituent being needed at C-3 for optimal activity. All of the synthetic analogues retained the original eleutherobin core and therefore provided no information about the importance of segments of the tricyclic core.
Eleutherobin represents one of five chemical structural types known to
arrest cells in mitosis by stabilizing microtubules. The other four are
paclitaxel, discodermolide, the epothilones, and the laulimalides
(39, 40, 41)
. Several pharmacophores have been proposed for
members of this group (42, 43, 44)
. The latter
(44)
included eleutherobin and proposed three regions of
common overlap between the chemotypes, shown as boxes A, B and C in
Fig. 4
. Region A of eleutherobin consists of the C-8 side chain, region
B encompasses the C-11 to C-13 segment of the tricyclic skeleton, and
region C consists of the C-15 substituent. The importance of regions A
and C is supported by the published structure-activity data for
eleutherobin analogues mentioned above (36, 37, 38)
. Our
demonstration that caribaeoside (9), which differs from
eleutherobin only in region B, shows a 200-fold lower activity
demonstrates an important role for this region in antimitotic activity.
Further studies will be required to determine whether the reduced
antimitotic activity of caribaeoside is attributable to reduced
affinity for tubulin and microtubules or to factors such as drug
uptake, extrusion, or metabolism.
Paclitaxel is an approved drug for the treatment of advanced ovarian cancer and metastatic breast cancer. It was originally isolated from Taxus brevifolia in 1971 (45) . Since then, over 350 related diterpenoids have been isolated from different species of the genus Taxus (46) , including compounds 11 and 12 described here. Compound 11 differs from paclitaxel in the nature of the N-acyl substituent on the C-13 phenylisoserine side chain and in the absence of the acetyl substituent at C-10. It was less active than paclitaxel, showing that the C-13 and C-10 substituents, although not essential for activity, contribute to the high potency of paclitaxel. Compound 12 further differs from paclitaxel by the presence of a ß-xylosyl substituent at C-7. Compound 12 was less active than compound 11, indicating that the C-7 substituent also contributes to the potency of paclitaxel.
Unexpected Outcomes.
An unexpected outcome of this study is that although the active
compounds we isolated belong to known antimitotic chemotypes, they were
found in organisms not known or suspected to produce them. To our
knowledge, rhizoxin compounds have previously only been isolated from
the rice seedling blight fungus Rhizopus chinensis and
unidentified species of the same genus (29)
. We have now
identified rhizoxin analogues in marine bacterial isolates of the genus
Pseudomonas, which is common in Pacific Northwest waters.
Eleutherobin was originally isolated from the soft coral
Eleutherobia sp. (possibly E. albiflora)
collected in Western Australia (23)
. We now identify
eleutherobin in the Caribbean octocoral Erythropodium
caribaeorum. This is of practical significance because it has not
been possible to obtain sufficient amounts of natural or synthetic
eleutherobin for preclinical development (47)
. The
taxonomic classification of this source was confirmed by the
identification of large quantities of the erythrolide diterpenoids
characteristic of this species (48)
. In contrast to
Eleutherobia, E. caribaeorum is widespread in the
Caribbean and Florida (49, 50, 51)
, abundant in certain areas,
and has been grown in aquaria. It may thus constitute a suitable source
of eleutherobin for preclinical and early phase clinical trials.
Paclitaxel and analogues have all been isolated from the bark of yew
trees (46)
, from endophytic fungi isolated from the
Taxus species or Taxodium distichum (52
, 53)
, and recently from an epiphytic fungus on the rubiaceous
plant Maguireothamnus speciosus (54)
. It was
surprising to find paclitaxel analogues in the bark of a
non-Taxus tree. The taxonomic classification of our extract
was confirmed by the presence of the triterpenoid glycosides
characteristic of the genus Ilex (55)
. It is
possible that an endophytic fungus is responsible for their production
in Ilex macrophylla.
Perhaps the most important outcome of this study is that the assay permitted us to detect antimitotic agents in extracts that were not found to contain them using other methods. E. caribaeorum has been subjected to extensive chemical characterization (48) , but eleutherobin compounds were not detected because they are very minor components. The COMPARE algorithm, which is able to detect similar differential patterns of growth inhibition for the 60 human cell lines in the NCI anticancer drug screen, has been used successfully to identify new antimitotic agents within the NCI chemical repository of pure compounds (13) . Extracts in the NCI Natural Products Repository have also been tested against the NCI cell line panel. A COMPARE analysis using paclitaxel as the probe compound identified 47 plant extracts with a Pearson correlation coefficient above 0.6 (not shown). Three of these extracts were positive in our antimitotic screen, and all three were from Catharanthus roseus, the plant from which the Vinca alkaloids were originally isolated (1) . The analysis did not identify the extract from Ilex macrophylla, the growth inhibition pattern of which does not resemble that of paclitaxel and other antimitotic compounds. This illustrates the usefulness of the cell-based assay for the identification of active compounds present in very low abundance in crude natural extracts. The assay should greatly facilitate the discovery and development of novel antimitotic agents and their characterization in the context of living cells.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Supported by the Canadian Breast Cancer Research
Initiative and the United States Department of Defense Breast Cancer
Research Program Idea Award DAMD17-99-1-9088 (to M. R.) and the
National Cancer Institute of Canada (to R. J. A.). 
2 To whom requests for reprints should be
addressed, at Department of Biochemistry and Molecular Biology,
University of British Columbia, 2146 Health Sciences Mall, Vancouver,
British Columbia, V6T 1Z3 Canada. Phone: (604) 822-2304; Fax:
(604) 822-5227; E-mail: michel{at}otter.biochem.ubc.ca
3 The abbreviations used are: NCI, National Cancer
Institute; ELICA, enzyme-linked immunocytochemical assay; HRP,
horseradish peroxidase.
Received 10/25/99. Accepted 7/19/00.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
-methane rearrangement. J. Am. Chem. Soc., 106: 5026-5027, 1984.
This article has been cited by other articles:
|
|
 |
|
  J. E. Loper, M. D. Henkels, B. T. Shaffer, F. A. Valeriote, and H. Gross Isolation and Identification of Rhizoxin Analogs from Pseudomonas fluorescens Pf-5 by Using a Genomic Mining Strategy Appl. Envir. Microbiol., May 15, 2008; 74(10): 3085 - 3093. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Fonrose, F. Ausseil, E. Soleilhac, V. Masson, B. David, I. Pouny, J.-C. Cintrat, B. Rousseau, C. Barette, G. Massiot, et al. Parthenolide Inhibits Tubulin Carboxypeptidase Activity Cancer Res., April 1, 2007; 67(7): 3371 - 3378. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Vassal, C. Barette, X. Fonrose, R. Dupont, E. Sans-Soleilhac, and L. Lafanechere Miniaturization and Validation of a Sensitive Multiparametric Cell-Based Assay for the Concomitant Detection of Microtubule-Destabilizing and Microtubule-Stabilizing Agents J Biomol Screen, June 1, 2006; 11(4): 377 - 389. [Abstract] [PDF]
|
|
|
|
 |
|
 D. Sampath, L. M. Greenberger, C. Beyer, M. Hari, H. Liu, M. Baxter, S. Yang, C. Rios, and C. Discafani Preclinical Pharmacologic Evaluation of MST-997, an Orally Active Taxane with Superior In vitro and In vivo Efficacy in Paclitaxel- and Docetaxel-Resistant Tumor Models. Clin. Cancer Res., June 1, 2006; 12(11): 3459 - 3469. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Jiang, B. Zhao, R. Britton, L. Y. Lim, D. Leong, J. S. Sanghera, B.-B. S. Zhou, E. Piers, R. J. Andersen, and M. Roberge Inhibition of Chk1 by the G2 DNA damage checkpoint inhibitor isogranulatimide Mol. Cancer Ther., October 1, 2004; 3(10): 1221 - 1227. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. DeBonis, D. A. Skoufias, L. Lebeau, R. Lopez, G. Robin, R. L. Margolis, R. H. Wade, and F. Kozielski In vitro screening for inhibitors of the human mitotic kinesin Eg5 with antimitotic and antitumor activities Mol. Cancer Ther., September 1, 2004; 3(9): 1079 - 1090. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
), or
ELICA (
). Experiments were carried out in triplicate, and values
indicate means; bars, SD.
