Affiliations of authors: M. R. Vredenburg, J. Veith, P. Pera, K. Kee, A. Sharma, P. Kanter, W. R. Greco, R. J. Bernacki, Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, NY; I. Ojima, State University at Stony Brook, NY; F. Cabral, The University of Texas Medical School, Houston.
Correspondence to: Ralph J. Bernacki, Ph.D., Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Sts., Buffalo, NY 14263 (e-mail: Ralph.Bernacki{at}roswellpark.org).
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ABSTRACT |
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INTRODUCTION |
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Although paclitaxel is the drug of choice for many chemotherapeutic regimens, its effectiveness is often limited because many tumors display drug resistance. Cells can acquire resistance to paclitaxel by at least two different mechanisms (18). First, alterations in either the assembly or stability of microtubules can lead to paclitaxel resistance (19). Second, overexpression of the mdr1 gene, which encodes P-glycoprotein (Pgp), can also confer resistance to paclitaxel. Pgp, which belongs to the adenosine triphosphate-binding cassette (ABC) transporter family of proteins (20), is located within the cell membrane where it functions as a xenobiotic pump that pumps many chemotherapeutic agents (e.g., paclitaxel) and numerous other natural products out of cells (21). Of the seven known ABC transporters involved in multidrug resistance, only Pgp confers cellular resistance to taxanes when it is overexpressed (22).
To improve paclitaxel's effectiveness, it is essential to overcome Pgp-mediated tumor resistance. We showed previously that several taxanes derived from 14-hydroxy-10-deacetylbaccatin III, including IDN-5109, are more effective than paclitaxel in inhibiting the growth of Pgp-expressing tumors and cell lines (2328). IDN-5109 induces a strong G2/M arrest and apoptosis (29) and is an effective growth-inhibitory agent when it was administered intravenously to mice bearing human tumor xenografts (30). Recently, IDN-5109 was also found to inhibit tumor growth when administered orally to mice bearing xenograft tumors derived from human tumor cells that were either sensitive or moderately resistant to paclitaxel (31,32).
On the basis of the reported ability of IDN-5109 to overcome Pgp-mediated drug resistance, we evaluated the growth-inhibitory properties of this novel taxane in Pgp-nonexpressing human tumor cell lines and in clones derived from those cell lines that express high levels of Pgp. We also compared the effects of IDN-5109, paclitaxel, and docetaxel on cell growth, tubulin polymerization, and the uptake and retention of the Pgp substrate, rhodamine 123 (Rh-123), by these cell lines. We next determined the intracellular dynamics of IDN-5109 and paclitaxel in human breast carcinoma cells that differed with respect to Pgp expression by use of radiolabeled derivatives of these taxanes. Finally, we examined the activity of IDN-5109 on paclitaxel-resistant, Pgp-expressing xenograft tumors derived from human colon carcinoma cells implanted in mice.
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MATERIALS AND METHODS |
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The following human carcinoma cell lines, none of which express Pgp (Pgp negative), were used: MDA435/LCC6 breast carcinoma cells (33), MCF-7 breast carcinoma cells (34), A2780 ovarian carcinoma cells (35), and HT-1080 sarcoma cells (36). We also used multidrug-resistant human tumor cell lines that had been derived from those drug-sensitive, Pgp-negative cell lines by exposing them to increasing levels of doxorubicin in culture. These drug-resistant cells included MCF-7Adr® (37) and A2780-DX5 (38) cells, both of which express Pgp, and HT-1080/DR4 cells, which express the multidrug resistance-related protein (MRP) and lung resistance protein (LRP) but not Pgp (39). MDA435/LCC6mdr1 cells, which express high levels of Pgp, were derived from MDA435/LCC6 cells that were transfected with a retrovirus engineered to constitutively express the mdr1 gene (33) and were provided by Dr. R. Clarke, Georgetown University Medical School, Washington, DC. The human colon carcinoma cell lines SW-620, DLD1, and HCT-15, which express low, intermediate, and high levels of Pgp, respectively, were also used (4042). All of the other human tumor cell lines were obtained from the Division of Cancer Treatment Tumor Repository, National Cancer Institute (Frederick, MD) or the American Type Culture Collection (Manassas, VA). Two animal cell linesChinese hamster ovary (CHO) cells and Tax-18 CHO cells, a paclitaxel-dependent mutant CHO cell line with impaired mitotic spindle assemblywere described previously (43). All of the cells were maintained at 37 °C in 5% CO2 in RPMI-1640 medium supplemented with 5% Nu-serum, 5% fetal bovine serum (Atlanta Biological, GA), 10 mM HEPES, and 2 mM L-glutamine (Life Technologies Inc. [GIBCO BRL], Rockville, MD).
Drugs and Reagents
Rh-123, supplied by Dr. Hans Minderman (Roswell Park Cancer Institute, Buffalo, NY) or purchased from Sigma Chemical Co. (St. Louis, MO), was diluted in water to give a 1-mg/mL stock solution. Doxorubicin and verapamil (Sigma Chemical Co.) were dissolved in 10% dimethyl sulfoxide (DMSO) and water, respectively, to give stock solutions of 4 mM each. For in vitro experiments, the Pgp drug resistance reversal agent, tRA96023 (SB-RA-31012), and taxane analogues 94004 (SB-T-10113), 94045 (SB-T-101141), 94042 (SB-T-10114), 95049 (SB-T-101171), and 95048 (SB-T-10117) were synthesized as described previously (2328). Each was then dissolved in DMSO at a concentration of 4 mM. Paclitaxel, docetaxel, IDN-5109, IDN-5102, IDN-5106, and IDN-5111 (supplied by Indena S.p.A., Milan, Italy) were also dissolved in DMSO at a concentration of 4 mM. [3H]Paclitaxel (specific activity, 2.6 Ci/mmol) was purchased from Moravek Biochemicals (Brea, CA). [3H]IDN-5109 (specific activity, 60 Ci/mmol) was synthesized by American Radiolabeled Chemicals, Inc. (St. Louis, MO).
In Vitro Growth-Inhibition Assay
The inhibitory effects of various drugs on cell growth were assessed by use of sulforhodamine B (SRB) (Sigma Chemical Co.), a dye-based assay, which indirectly determines cell number by measuring membrane-associated proteins, as described previously (44). Briefly, 1 x 105 exponentially proliferating cells were seeded on 96-well microtiter plates in complete growth medium and incubated at 37 °C for 1518 hours to allow the cells to attach to the substrate before taxanes were added. For each taxane tested, five 96-well microtiter plates were screened in parallel. All cell lines were exposed to 1012 different concentrations of each drug, covering a 5- to 6-log range of concentrations, at 37 °C, 5% CO2. MCF-7, MCF-7R, MDA435/LCC6, MDA435/LCC6mdr1, A2780, A2780DX5, HT1080, SW-620, DLD1, and HCT-15 cells were exposed to drugs for 72 hours (approximately 3.3 cell doublings), whereas HT1080/DR4 cells were exposed to drugs for 100 hours (approximately 3.5 cell doublings). Cells were fixed in situ for 1 hour at 4 °C with ice-cold 50% trichloroacetic acid. The plates were then washed six times with water, and 150 µL of 0.4% SRB was added to each well. After a 5-minute incubation at room temperature, the plates were rinsed with 0.1% acetic acid and air-dried. Bound SRB was solubilized by adding a 100 µL 10 mM Tris base (pH 10.5) per well and was allowed to stand at room temperature for 5 minutes. The optical density (OD) of each well was measured at 570 nm. Under these conditions, cell number is proportional to OD. We determined the concentration of each drug that inhibited 50% of cell growth (IC50) using empirically determined concentrations of drugs between 10 pM to 30 mM. The IC50 was obtained by plotting a concentrationeffect curve as described in the "Statistical Methods" section. A minimum of three separate experiments was used to derive the IC50 values presented. The R/S ratio, which is a measure of cellular resistance to a particular chemotherapeutic agent, was calculated by dividing the IC50 obtained for a drug-resistant cell line by the IC50 of the corresponding drug-sensitive cell line (e.g., MCF-7R IC50/MCF-7 IC50).
Colony-Forming Assay
Equal numbers (1 x 105) of CHO and TAX-18 CHO cells were seeded in 24-well plates in complete growth medium containing various concentrations of paclitaxel or IDN-5109 and incubated at 37 °C in 5% CO2 for 6 days until visible colonies formed. Cells were then stained with 0.05% methylene blue, as described previously (43), to visualize the number of viable colonies and were photographed.
Tubulin Polymerization Assay
Temperature-induced microtubule assembly or disassembly in vitro was assayed as described previously (45,46). Briefly, bovine brain tubulin (Cytoskeleton, Inc., Denver, CO) at 10 mg/mL in G-PEM buffer (i.e., 80 mM piperazine 1,4-diethane sulfonic acid, 0.5 mM MgCl2, 1 mM EGTA, and 1 mM guanosine 5'-triphosphate) was thawed on ice and diluted to 0.7 mg/mL in G-PEM buffer. To the tubulin solution, either paclitaxel or IDN-5109 in DMSO was added, bringing the final concentration of taxanes to 10 µM in 2% DMSO. Samples were mixed by vortexing and were placed on ice. Duplicate samples were incubated in a 32 °C water bath, and the absorbance of each sample at 340 nm, indicating tubulin polymerization, was measured every 30 seconds for 20 minutes by use of a Beckman DU640 spectrophotometer (Beckman Instruments Inc., Fullerton, CA). The absorbance readings for duplicate samples were averaged, and the background was subtracted.
Measuring Cellular Accumulation of [3H]Paclitaxel and [3H]IDN-5109
Exponentially growing MDA435/LCC6 or MDA435/LCC6mdr1 cells (1 x 105) were seeded into 24-well tissue culture plates and incubated at 37 °C in 5% CO2 for 12 hours to allow for adherence. [3H]Paclitaxel and [3H]IDN-5109 were diluted in RPMI-1640 growth medium that contained or lacked tRA96023. Cells were washed once with phosphate-buffered saline (PBS), and the diluted radiolabeled drugs were added to the cells at a concentration of 0.20.6 µCi/mL per well. After a 2-hour incubation at 37 °C, adherent cells were carefully washed twice with PBS, fresh medium containing or lacking tRA96023 was added, and the cells were returned to the incubator to allow the efflux of radiolabeled drugs. At 2-, 4-, 8-, and 10-hour efflux time points (i.e., after the addition of fresh medium), the medium was removed from the well corresponding to that time point and the cells were washed twice with PBS and then solubilized in 200 µL 1 N NaOH at 37 °C for 30 minutes. Aliquots (35 µL) of solubilized cells were removed for protein quantification by use of the Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Then 1 N HCl (150 µL) was added to a separate 150-µL aliquot of solubilized cells, and total radioactivity (in counts per minute [cpm]) was measured by use of a Model LS-6500 scintillation counter (Beckman Instruments, Wakefield, MA). Drug accumulation is expressed as counts per minute per milligram protein.
Measurement of Rh-123 and Doxorubicin Uptake and Efflux by Flow Cytometry
Aliquots of approximately 1 x 106 tumor cells in complete growth medium were placed into 5-mL polystyrene tubes. A solution of 5 µg/mL Rh-123 or 10 µM doxorubicin containing IDN-5109, tRA96023, paclitaxel, docetaxel, verapamil, or 94004 at 1 µM was added to the cells, which were then incubated at 37 °C for 1 hour (for Rh-123-containing additions) for 2 hours (for doxorubicin-containing additions). After this incubation, the cells were washed with PBS, and fresh medium containing only the original modulator (i.e., IDN-5109, tRA96023, paclitaxel, docetaxel, verapamil, or 94004) at 1 µM was added to the cells. The cells were incubated at 37 °C for 4 hours to allow the efflux of Rh-123 or doxorubicin. The cells were then washed and resuspended in ice-cold PBS and subjected to flow cytometry to analyze the intracellular levels of Rh-123 and doxorubicin.
Flow cytometry was carried out as described previously (4753) by use of a FACSCAN flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA) interfaced with Macintosh Cell Quest software (Becton Dickinson Immunocytometry Systems). Briefly, cells incubated with Rh-123 and doxorubicin were excited with a single-beam argon laser running at 15-mW output. Rh-123 fluorescent emission was collected through a 530/30-nm band-pass filter, and doxorubicin fluorescent emission was collected though a 680/20-nm long-pass filter, with photomultiplier pulses logarithmically amplified in both cases. Cells were selected for analyses based on their forward-angle scatter and right-angle scatter. The resulting data were analyzed with Winlist software (Verity Software House, Torpsham, ME). Fluorescence intensities of drug-treated cells were quantified by use of arbitrary units and were compared with those of control cells treated with the specific vehicle for each drug evaluated (i.e., DMSO, for taxanes; water, for verapamil; and 10% DMSO, for doxorubicin).
Animals and Tumor Xenografts
Female athymic nude (nu/nu) mice, 610 weeks of age and weighing 2025 g, were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and housed at the Medical Research Complex at Roswell Park Cancer Institute. Animal care complied with the Institutional Animal Care and Use Committee guidelines. SW-620 or DLD1 human tumor cells were harvested by trypsinization, washed twice in ice-cold PBS, and adjusted to 12 x 107 viable cells/mL by use of trypan blue dye exclusion as the criterion for viability. Then 0.2 mL of each cell suspension was injected subcutaneously into the right flank of each mouse (54,55). After the tumors reached a palpable size of 50100 mm3, the mice (in groups of five each) were given four separate doses (either by the oral route or by intravenous injection at 4-day intervals) of each drug. Oral paclitaxel treatment began on day 7, intravenous paclitaxel treatment began on day 10, oral IDN-5109 treatment began on day 11, and intravenous IDN-5109 treatment began on day 13 after tumor cell injection. The experiment concluded on day 150 after tumor cell injection.
Drug Preparation for In Vivo Experiments
Paclitaxel was prepared as a 6-mg/mL stock solution in equal parts of Cremophor ELP (BASF, Ludwigshafen, Germany) and absolute ethanol. IDN-5109 was prepared as a 30-mg/mL stock solution in equal parts of Tween 80 (polyoxyethylenesorbitan monooleate; from Sigma Chemical Co.) and absolute ethanol. Each stock solution was diluted further immediately before use in 0.9% NaCl (saline) so that the appropriate concentration of each drug could be injected in a volume of approximately 0.4 mL for a 20-g mouse. tRA96023 was prepared as a 10-mg/mL stock solution in equal parts of Cremophor ELP and absolute ethanol and diluted to 1 mg/mL in saline before use.
Statistical Methods
In vitro cell growth-inhibition assay.
The four-parameter Hill model (shown in equation 1) was used as the structural model for the concentrationeffect curve of each single agent (56,57). Equation 1
was fitted to the experimental data with iteratively reweighted nonlinear regression, with the estimation of the four parameters and accompanying standard errors. In equation 1
, E is the measured effect, C is the concentration of drug, Econ is the control response at zero drug concentration, B is the background response at infinite drug concentration, IC50 is the concentration of drug inducing a 50% inhibition of the maximal possible cell growth (Econ B), and m is the slope parameter of the concentrationeffect curve. When m is positive, the concentrationeffect curve rises with increasing agent concentration; when m is negative, the curve falls with increasing concentration. As the absolute value of m increases, the curve becomes steeper (on linearlog coordinates). The weighting factor used was the reciprocal of the square of the predicted response. This assumes a constant coefficient of variation for the data error structure, which is a common and reasonable assumption (56,57).
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MicroSoft FORTRAN, Microsoft, Bothell, WA, was used to develop the nonlinear curve-fitting software. The nonlinear regression procedure was coded with the Nash (58) version of the Marquardt algorithm.
In vivo tumor growth assay. For each animal, the tumor length (l) and width (w), each in mm, were measured by use of electronic calipers and recorded every 34 days. Tumor volume (v), in mm3, was calculated by use of the following formula: v = 0.4 (l x w2). The time in days to the predetermined target tumor volume of 600 mm3 was linearly interpolated from a plot of log(volume) versus time. Statistically significant differences in tumor volumes between control and drug-treated mice were determined by the CoxMantel test (59). For the CoxMantel test, the time-to-event data for animals that did not reach the target tumor volume, either because of long-term cure (defined as those animals that were still alive at the conclusion of the experiment whose tumors either completely regressed or did not reach the preset target volume) or early death because of drug toxicity, were treated as censored data. All statistical tests were two-sided.
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RESULTS |
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To begin to investigate the mechanism of action of IDN-5109, we first compared the growth-inhibitory properties of various taxanes in drug-sensitive cell lines and in drug-resistant cell lines that were derived from them. For three of these pairs of cell lines, the drug-resistant line (i.e., MCF-7Adr, MDA435/LCC6mdr1, or A2780-DX5) expresses Pgp. For the remaining pair of cell lines, of which neither cell line expresses Pgp, the drug-resistant line, HT-1080/DR4, expresses MRP and LRP, two other membrane transporters believed to confer cellular multidrug resistance. Fig. 1 shows the structures of paclitaxel, docetaxel, and the various taxane analogues used in this study. Minor alterations to the chemical structure of paclitaxel were associated with a decrease in the concentration of drug necessary to cause a 50% inhibition of growth (IC50) for a variety of Pgp-expressing, multidrug-resistant cell lines (Table 1
). However, such alterations had minimal effects on growth inhibition for the corresponding drug-sensitive cell lines that did not express Pgp. For example, lower concentrations of taxanes that have an acetyl group at the C-10 position of the moiety designated R5 in Fig. 1
(such as IDN-5109, IDN-94045, and IDN-95049) were required to inhibit the growth of drug-resistant cell lines that express Pgp than were required of taxanes that have a hydroxyl group at this position (such as 94004, 94042, and 95048).
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Effect of IDN-5109 on Tubulin Polymerization and Cell Growth
Because paclitaxel promotes the polymerization of tubulin both in vivo and in cell-free systems, we tested whether IDN-5109 was more effective than paclitaxel in stabilizing tubulin and promoting its polymerization. Both IDN-5109 and paclitaxel gave similar absorbance profiles in a temperature-dependent tubulin polymerization assay, suggesting that enhanced tubulin stabilization and/or polymerization could not account for greater growth-inhibitory activity of IDN-5109 compared with paclitaxel (Fig. 2, A). Further support for this conclusion came from the observation that CHO-TAX-18 cells, which express a mutant form of tubulin that causes impaired mitotic spindle assembly and normally require low levels of paclitaxel for growth, required 20- to 40-fold lower concentrations of IDN-5109 than paclitaxel for growth (Fig. 2
, B). This finding suggests that, in these cells, IDN-5109 is more potent in its ability to sustain cell growth and/or that its uptake and/or retention by these cells is increased. Moreover, the growth of both wild-type CHO cells and mutant CHO-TAX-18 cells was inhibited by IDN-5109 concentrations 10- to 20-fold lower than the concentrations of paclitaxel required to inhibit the growth of these cells. In contrast, the taxane-based drug resistance reversal agent tRA96023, which lacks the C-13 side chain necessary for taxanetubulin interaction, had no effect on tubulin polymerization (Fig. 2
, A) or on cell proliferation (data not shown) when present at 30 mM, its maximal soluble concentration in tissue culture.
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The superior growth-inhibitory effect of IDN-5109 in Pgp-expressing tumor cell lines, as compared with paclitaxel, could be because it is a poor substrate for Pgp, as was suggested previously (2932) or because it binds to Pgp, blocking the action of the pump and, consequently, its own efflux from the cell. We tested the latter possibility by measuring the ability of IDN-5109 to modulate Pgp activity. Specifically, we used flow cytometry to measure the accumulation of Rh-123, a fluorescent Pgp substrate, in three human colon carcinoma cell lines, SW620, DLD1, and HCT-15, that express increasing amounts of the messenger RNA (mRNA) that encodes Pgp, respectively (60). The cells were incubated with Rh-123 for a brief uptake period, then washed and incubated in the absence of Rh-123 for a 4-hour efflux period (Fig. 3, A). Flow cytometry was used to generate histograms representing Rh-123 fluorescence within a population of cells. Separate histograms were generated for each cell line/treatment group before incubation with Rh-123 (autofluorescence), immediately after incubation with Rh-123 (uptake), and after the 4-hour efflux period (uptake and efflux). Those histograms were then superimposed to generate one histogram per drug treatment group for each cell line. For each of these vehicle-treated, Pgp-expressing colon tumor cell lines, the intracellular levels of Rh-123 decreased during the efflux period, as demonstrated by the substantial numbers of cells having fewer than 1000 arbitrary units of fluorescence intensity (Fig. 3
, A). The multiple peaks of fluorescence intensity displayed by the vehicle-treated DLD1 and SW620 cells after the efflux period suggest that these cell lines contain a mixed population of cells with different amounts of mdr1 mRNA. In all three cell lines, the addition of the Pgp modulator verapamil during the uptake and efflux periods led to an increase in the uptake and retention of Rh-123. However, cells exposed to either tRA96023 or IDN-5109 accumulated and retained more Rh-123 than did cells treated with either vehicle or verapamil. Surprisingly, the toxic taxane IDN-5109 was nearly as potent as the nontoxic multidrug resistance reversal agent tRA96023 in increasing Rh-123 retention, demonstrating that IDN-5109 shows a strong inhibitory effect on the activity of Pgp. In contrast, cells treated with either paclitaxel or docetaxel accumulated and retained only slightly higher levels of Rh-123 than did cells treated with vehicle, and they accumulated and retained much less Rh-123 than did cells treated with IDN-5109 or tRA96023. Among the three multidrug-resistant colon carcinoma cell lines, tRA96023 and IDN-5109 blocked the efflux of Rh-123 more effectively in SW620 cells, which express the lowest amount of mdr1 mRNA, than in HCT-15 cells, which express the highest amount of mdr1 mRNA. From these results, we conclude that IDN-5109 modulates the activity of Pgp, resulting in the greater intracellular uptake and retention of the Pgp substrate, Rh-123, as compared with either paclitaxel or docetaxel.
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Doxorubicin, another Pgp substrate, is a standard treatment of breast cancer. We, therefore, measured the effects of tRA96023 and IDN-5109 on doxorubicin uptake and retention in MDA435/LCC6 and MDA435/LCC6mdr1 breast tumor cell lines. Cells treated with tRA96023 and IDN-5109 accumulated and retained higher levels of doxorubicin than cells treated with verapamil, paclitaxel, or docetaxel, providing further evidence that these two taxanes can modulate Pgp activity (Fig. 3, B). More specifically, based on integration of the doxorubicin fluorescence peak, the addition of 1 µM tRA965023 or IDN-5109 increased MDA435/LLC6mdr1 cellular retention of doxorubicin 1.9- and 1.7-fold, respectively, in comparison to paclitaxel.
Because both tRA96023 and IDN-5109 were effective in maintaining high intracellular concentrations of Pgp substrates in cells that expressed high levels of Pgp, we tested whether these drugs if used in combination with each other or with other taxanes would have synergistic effects. MDA435/LCC6mdr1 cells treated simultaneously with tRA96023 and either IDN-5109, paclitaxel, or docetaxel retained more Rh-123 than cells treated with each agent alone (Fig. 4). As expected, cells treated with the combination of IDN-5109 and tRA96023 retained the most Rh-123, suggesting that this combination of drugs could be more effective against Pgp-expressing tumors than IDN-5109 alone.
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We compared the uptake and retention of IDN-5109 and paclitaxel by use of radiolabeled derivatives of each drug to ascertain whether the observed inhibition of Pgp activity noted with IDN-5109 was associated with increased intracellular levels of IDN-5109 in Pgp-expressing cells. We measured the radioactivity in tumor cells that were incubated for 2 hours in the presence of [3H]paclitaxel and [3H]IDN-5109 and then in fresh medium lacking the radiolabeled compounds for up to 10 hours. Both [3H]paclitaxel and [3H]IDN-5109 accumulated to similarly high levels in MDA435/LCC6 cells, which have no detectable Pgp expression (Fig. 5). In contrast, Pgp-expressing MDA435/LCC6mdr1 cells accumulated substantially more [3H]IDN-5109 than [3H]paclitaxel, but the levels of [3H]IDN-5109 did not approach those observed to accumulate in MDA435/LCC6 cells. This suggests that the ability of IDN-5109 to decrease Pgp activity may result in its increased retention by Pgp-expressing cells compared with the retention of paclitaxel, which does not modulate Pgp activity. After the 2-hour uptake period, MDA435/LCC6mdr1 cells had accumulated 10-fold lower intracellular concentrations of [3H]paclitaxel than MDA435/LCC6 cells and, after 2 hours of efflux, [3H]paclitaxel was eliminated completely from the Pgp-expressing cells but not from the Pgp-nonexpressing cells.
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Effects of IDN-5109 and Paclitaxel on Xenograft Tumors Derived From Human Colon Cancer Cells
Because the in vitro results suggested that IDN-5109 might be an effective chemotherapeutic agent in vivo, we tested IDN-5109 in a mouse xenograft model of tumors derived from the subcutaneous injection of SW-620 human colon carcinoma cells. SW-620 cells were chosen for this study because they expressed sufficient levels of Pgp to allow rapid efflux of Rh-123 (Fig. 3, A), despite having the lowest level of Pgp mRNA of the three colon carcinoma cell lines studied. Intrinsic Pgp expression in the gut limits paclitaxel uptake and, thus, its bioavailability, when administered orally. However, we considered the possibility that IDN-5109 might have better oral bioavailability than paclitaxel because we had observed the inhibitory effects of IDN-5109 on Pgp activity in vitro (Fig. 3
). We, therefore, compared the chemotherapeutic activities of IDN-5109 and paclitaxel administered either orally or intravenously to mice bearing xenograft tumors derived from SW-620 cells. Table 2
shows that established SW-620 tumors were resistant to paclitaxel administered intravenously at its maximum tolerated cumulative dose of 100 mg/kg. In contrast, IDN-5109 administered intravenously at a cumulative dose of 240 mg/kg was effective in retarding the growth of tumors derived from SW-620 cells, resulting in a 79-day delay (95% confidence interval [CI] = 51 to 107 days) in tumor growth compared with the growth of tumors in control animals. One animal that received intravenous IDN-5109 was considered to be cured because its tumor completely regressed and no tumor was palpable at the conclusion of the experiment on day 150 after tumor cell implantation.
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To improve on the antitumor effect demonstrated by IDN-5109 alone, we compared the effects of orally administered IDN-5109 given with and without the taxane-reversal agent, tRA96023, on the growth of xenograft tumors derived from paclitaxel-resistant DLD1 human colon carcinoma cells. DLD1 cells express approximately 7.6-fold more Pgp mRNA than SW-620 cells (60) and, presumably, more Pgp. When given at its maximum tolerated cumulative dose, paclitaxel did not inhibit the growth of tumors derived from DLD1 cells (data not shown). In contrast, mice that received IDN-5109 administered orally at a cumulative dose of 360 mg/kg had a statistically significant increase (P = .002) in the number of days (23 days; 95% CI = 15.2 to 30.8 days) that tumor growth was delayed compared with control mice that received the Tween vehicle (Table 3). Mice that received lower cumulative doses of IDN-5109 (120 or 240 mg/kg) administered orally had little or no delay in the growth of their tumors compared with control mice that received vehicle. However, mice that received the combination of IDN-5109 at 240 mg/kg and tRA96023 at 80 mg/kg had a statistically significant delay (P = .002) in the growth of their tumors (26 days; 95% CI = 18 to 70 days) compared with control mice that received the combination of Tween and vehicles. Of interest, increasing the dose of tRA96023 to 120 mg/kg in this combination treatment decreased the number of days that tumor growth was delayed to 10 days (95% CI = 5.8 to 14.2 days). Mice treated with the combination of IDN-5109 at 360 mg/kg and tRA96023 at 40 mg/kg had the longest delay in tumor growth (29 days; 95% CI = 20.6 to 37.4 days). All of the animals treated with the various drug combinations exhibited minimal weight loss (Table 3
). These results suggest that the combination of IDN-5109 and the nontoxic reversal agent tRA96023 may be an effective treatment to inhibit or delay the growth of tumors that express higher levels of Pgp.
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DISCUSSION |
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IDN-5109 is a semisynthetic taxane that is a highly effective growth-inhibitory agent against paclitaxel-resistant tumor cell lines in vitro (24) and human tumor xenografts in vivo (30,31). IDN-5109 also demonstrated improved growth-inhibitory properties compared with paclitaxel in two xenograft tumor models derived from cells that were moderately responsive to paclitaxel (32). Although a preliminary study (29) has indicated that IDN-5109 induces a strong G2/M arrest and induces apoptosis in Pgp-expressing CEM VBLr cells, the mechanism(s) responsible for the increased activity of IDN-5109 in paclitaxel-resistant, Pgp-expressing tumors has remained a mystery.
Our findings from in vitro growth-inhibition assays confirm that IDN-5109 is a novel taxane that is effective against human tumor cell lines that are resistant to paclitaxel and docetaxel. However, differences in chemical structure confer on IDN-5109 an advantage, relative to paclitaxel, against Pgp-expressing tumors (24). This study sought to understand why IDN-5109 is much more effective than paclitaxel at inhibiting the growth of Pgp-expressing human tumor cells. Increased activity of IDN-5109 could be explained by numerous possibilities, including increased cellular uptake, enhanced apoptotic induction, enhanced tubulin polymerization capabilities, and/or increased cellular retention of drug.
It is unlikely that IDN-5109's enhanced growth-inhibitory properties, as compared with the growth-inhibitory properties of paclitaxel, against Pgp-expressing cells is due to direct effects on microtubule stability. We have shown that IDN-5109 and paclitaxel have similar effects on tubulin polymerization in vitro and on colony formation in vivo by use of the tubulin mutant-containing Tax-18 CHO cell line. These results suggest that the differences in molecular mechanism(s) of action between paclitaxel and IDN-5109 involve a target other than tubulin.
It was suggested previously that IDN-5109 is not a substrate for Pgp, since IDN-5109 is active in Pgp-expressing cell lines (29). In this study, we explored an alternative possibility, that the modulation of Pgp activity, as measured by the accumulation and retention of various Pgp substrates by tumor cells that express different amounts of Pgp, could represent a potential mechanism to explain the superior growth-inhibitory activity of IDN-5109 against Pgp-expressing tumors. Our data suggest that, in Pgp-expressing cells, IDN-5109 modulates Pgp action, as demonstrated by increased levels of Pgp substrate (Rh-123 and doxorubicin) retention in these cells. The acetyl group at the C-10 position of the taxane nucleus appears to be important in conferring IDN-5109 with increased Pgp modulation characteristics. This structural feature may explain the enhanced in vitro activity of taxanes with an acetyl group at this position, as compared with taxanes containing a hydroxyl group at the C-10 position.
Our current model for IDN-5109's mechanism of action involves IDN-5109 binding to Pgp and inhibiting the efflux activity of the pump, thus allowing IDN-5109 and other compounds to accumulate in cells. This would explain the superior antitumor effect of IDN-5109, compared with that of paclitaxel, on tumors that express Pgp. The results demonstrating increased intracellular accumulation and retention of [3H]IDN-5109 versus [3H]paclitaxel in a Pgp-expressing human breast carcinoma cell line are consistent with increased tumor growth inhibition of Pgp-expressing tumor cell lines by IDN-5109 as compared with paclitaxel. [3H]IDN-5109 was retained in MDA435/LCC6mdr1 cells for up to 10 hours, while paclitaxel was effluxed rapidly from the cells. These findings lend support to our hypothesis that IDN-5109, by directly affecting Pgp action, is a self-modulating agent capable of blocking its own efflux and increasing its accumulation in Pgp-expressing tumor cells. Future studies will explore the physical nature of the interaction of IDN-5109 with Pgp. The ability of IDN-5109 to inhibit the efflux action of Pgp opens possibilities for synergistic interaction between IDN-5109 and other drugs (e.g., doxorubicin) that demonstrate resistance in Pgp-positive, multidrug-resistant tumors.
The taxane-based drug resistance reversal agent tRA96023 also modulated Pgp pump activity, and the combination of IDN-5109 and tRA96023 appeared to increase inhibition of Pgp activity, allowing for an increase in retention of Pgp substrates and increased tumor cell growth inhibition. The use of this nontoxic, drug resistance reversal agent with other Pgp-excludable drugs (e.g., doxorubicin and vinca alkaloids) will be explored in future studies. An increase in antitumor activity with such drug combinations against Pgp-expressing tumors is a distinct possibility, especially considering the results demonstrating tRA96023's ability to enhance retention of doxorubicin, [3H]paclitaxel, and [3H]IDN-5109.
A previous study (31) demonstrated that IDN-5109 is an orally bioavailable taxane. In this study, we compared the antitumor effects of IDN-5109 and paclitaxel, using both intravenous and oral administration, in nude mice bearing tumor xenografts derived from Pgp-expressing cells. Experimental therapeutic studies in mice strongly indicated that IDN-5109 when administered either intravenously or orally is superior to paclitaxel in inhibiting the growth of paclitaxel-resistant tumors when administered either intravenously or orally. The increased oral bioavailability of IDN-5109, as compared with paclitaxel, may be a result of modulatory effects by IDN-5109 on the action of the Pgp pump inherently expressed in the gut. Of interest, lower doses (320 and 480 mg/kg) of orally administered IDN-5109 were more effective against tumors than a higher dose (720 mg/kg). This result could reflect experimental error associated with the small test groups of mice used (n = 5) for these studies. Alternatively, IDN-5109 uptake from the gut into the bloodstream may have been affected by the increase in gut toxicity elicited by higher doses of drug. Nevertheless, we observed that all of the oral doses of IDN-5109 described herein demonstrated an antitumor effect. It is also important to note that oral doses of IDN-5109 equaling intravenous doses (240 mg/kg) showed little tumor-growth inhibition against Pgp-expressing SW-620 colon tumor xenografts in mice. When administered orally, paclitaxel showed little antitumor activity suggesting that it is excluded from the bloodstream by the natural expression of the Pgp pump in the intestinal lining. IDN-5109 formulated in Tween 80/ethanol proved to be less toxic than paclitaxel formulated in Cremaphor/ethanol, allowing for the administration of higher doses of IDN-5109 to mice.
The combination of IDN-5109 with tRA96023 was more effective that IDN-5109 alone against tumors derived from paclitaxel-resistant DLD1 colon carcinoma cells, suggesting that the combination of the two agents may be of therapeutic value. The in vitro flow cytometric data suggested that the combination's enhanced activity as compared with each agent alone might be due to increased intracellular drug retention within the tumor cell. The combination of 240 mg/kg of IDN-5109 with 80 mg/kg of tRA96023 produced a statistically significant (P = .006) antitumor effect in vivo compared with IDN-5109 alone. Of interest, increasing the dose of tRA96023 to 120 mg/kg in combination with IDN-5109 produced no statistically significant difference in tumor-growth inhibition compared with IDN-5109 alone. This finding may suggest that, depending on the dose, tRA96023 can either increase IDN-5109 uptake when it is administered orally or, at high doses, can compete for IDN-5109 uptake, thus compromising the oral bioavailability of IDN-5109. Pharmacokinetic studies are necessary to better understand how tRA96023 acts when given in combination with IDN-5109.
Preclinical toxicology studies conducted at the Roswell Park Cancer Institute indicated that the toxicity profile of IDN-5109 in rats and dogs was similar to the toxicity profiles of other clinically useful, antineoplastic agents (Kanter P: unpublished results). In both species, gastrointestinal toxicity was the dose-limiting toxicity. Myelosuppression, primarily decreased neutrophils, was observed at high doses of IDN-5109, with little observed effect on platelets. Other toxic effects included anorexia and weight loss, hind-limb weakness (rats), hair loss, diarrhea, and vomiting. Neurologic toxicity was also noted in rats but was reversible. No neurotoxicity was evident in dogs. IDN-5109's effectiveness against drug-resistant tumors, as demonstrated here and in preclinical testing, has facilitated its entrance into clinical trial, where it may prove to be more effective than paclitaxel or docetaxel against a variety of tumors, especially those exhibiting a multidrug resistance phenotype. Currently, drug therapies for colon cancer treatment are severely limited. IDN-5109 may be able to broaden the spectrum of taxane use to include colon tumors, thus providing an alternative to the current regimens of treatment.
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NOTES |
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We acknowledge the many helpful suggestions and support of Dr. E. Mihich, Director, Grace Cancer Drug Center, and Dr. C. Stewart, Director, Flow Cytometry, Roswell Park Cancer Institute, Buffalo, NY, and Dr. E. Bombardelli, Scientific Director, Indena, S.p.A., Milan, Italy.
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REFERENCES |
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1
Rowinsky EK, Donehower RC. Paclitaxel (Taxol). N Engl J Med 1995;332:100414.
2 Eisenhauer EA, Vermorken JB. The taxoids. Comparative clinical pharmacology and therapeutic potential. Drugs 1998;55:530.[Medline]
3 Wiseman LR, Spencer CM. Paclitaxel: an update of its use in the treatment of metastatic breast cancer and ovarian and other gynecological cancers. Drugs Aging 1998;12:30534.[Medline]
4 McGuire WP, Rowinsky EK, Rosenshein NB, Grumbine FC, Ettinger DS, Armstrong DK, et al. Taxol: a unique antineoplastic agent with significant activity in advanced ovarian epithelial neoplasms. Ann Intern Med 1989;111:2739.[Medline]
5 Liebmann JE, Cook JA, Lipschultz C, Teague D, Fisher J, Mitchell JB. Cytotoxic studies of paclitaxel (Taxol) in human tumor cell lines. Br J Cancer 1993;68:11049.[Medline]
6 Jordan MA, Toso RJ, Thrower D, Wilson L. Mechanism of mitotic block and inhibition of cell proliferation by Taxol at low concentrations. Proc Natl Acad Sci U S A 1993;90:95526.[Abstract]
7
Kumar N. Taxol-induced polymerization of purified tubulin. Mechanism of action. J Biol Chem 1981;256:1043541.
8 Schiff PB, Fant J, Horwitz SB. Promotion of microtubule assembly in vitro by Taxol. Nature 1979;277:6657.[Medline]
9 Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980;77:15615.[Abstract]
10
Parness J, Horwitz SB. Taxol binds to polymerized tubulin in vitro. J Cell Biol 1981;91(2 Pt 1):47987.
11 Schiff PB, Horwitz SB. Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins. Biochemistry 1981;20:324752.[Medline]
12 Manfredi JJ, Parness J, Horwitz SB. Taxol binds to cellular microtubules. J Cell Biol 1982;94:68896.[Abstract]
13 Woods CM, Zhu J, McQueney PA, Bollag D, Lazarides E. Taxol-induced mitotic block triggers rapid onset of a p53 independent apoptotic pathway. Mol Med 1995;1:50626.[Medline]
14 Ireland CM, Pittman SM. Tubulin alterations in Taxol-induced apoptosis parallel those observed with other drugs. Biochem Pharmacol 1995;49:14919.[Medline]
15 Cheng L, Zheng S, Raghunathan K, Priest DG, Willingham MC, Norris JS, et al. Characterizations of paclitaxel-induced apoptosis and altered gene expression in human breast cancer cells. Cell Pharmacol 1995;2:24957.
16 Fan W. Possible mechanism of paclitaxel-induced apoptosis. Biochem Pharmacol 1999;57:121521.[Medline]
17 Blagosklonny MV, Fojo T. Molecular effects of paclitaxel: myths and reality. Int J Cancer 1999;83:1516.[Medline]
18 Casazza AM, Fairchild CR. Paclitaxel (Taxol): mechanisms of resistance. Cancer Treat Res 1996;87:14971.[Medline]
19
Schibler M, Cabral F. Taxol-dependent mutants of Chinese hamster ovary cells with alterations in - and
-tubulin. J Cell Biol 1986;102:152231.[Abstract]
20 Ueda K, Cardarelli C, Gottesman MM, Pastan I. Expression of a full-length cDNA for the human "MDR1" gene confers resistance to colchicine, doxorubicin, and vinblastine. Proc Natl Acad Sci U S A 1987;84:30048.[Abstract]
21 Bhalla K, Huang Y, Tang C, Self S, Ray S, Mahoney ME, et al. Characterization of a human myeloid leukemia cell line highly resistant to Taxol. Leukemia 1994;8:46575.[Medline]
22 Beck WT. Mechanisms of multidrug resistance in human tumor cells. The roles of P-glycoprotein, DNA topisomerase II, and other factors. Cancer Treat Rev 1990;17:1120.[Medline]
23 Ojima I, Slater JC, Michaud E, Kuduk SD, Bounaud PY, Vrignaud P, et al. Syntheses and structureactivity relationships of the second-generation antitumor taxoids: exceptional activity against drug-resistant cancer cells. J Med Chem 1996;39:388996.[Medline]
24
Ojima I, Slater JC, Kuduk SD, Takeuchi CS, Gimi RH, Sun CM, et al. Syntheses and structureactivity relationships of taxoids derived from 14-hydroxy-10-deacetylbaccatin III. J Med Chem 1997;40:26778.[Medline]
25 Ojima I, Kuduk SD, Pera P, Veith JM, Bernacki RJ. Synthesis and structureactivity relationships of nonaromatic taxoids: effects of alkyl and alkenyl ester groups on cytotoxicity. J Med Chem 1997;40:27985.[Medline]
26 Ojima I, Bounaud PY, Takeuchi C, Pera P, Bernacki RJ. New taxanes as highly efficient reversal agents for multidrug resistance in cancer cells. Bioorg Med Chem Lett 1998;8:18994.[Medline]
27 Ojima I, Bounaud PY, Bernacki RJ. New weapons in the fight against cancer. CHEMTECH 1998;28:316.
28 Ojima I, Bounaud PY, Bernacki RJ. Designing taxanes to treat multidrug-resistant tumors. Mod Drug Disc 1999;02:4552.
29
Distefano M, Scambia G, Ferlini C, Gaggini C, De Vincenzo R, Riva A, et al. Anti-proliferative activity of a new class of taxanes (14-hydroxy-10-deacetylbaccatin III derivatives) on multidrug-resistance-positive human cancer cells. Int J Cancer 1997;72:84450.[Medline]
30
Polizzi D, Pratesi G, Tortoreto M, Supino R, Riva A, Bombardelli E, et al. A novel taxane with improved tolerability and therapeutic activity in a panel of human tumor xenografts. Cancer Res 1999;59:103640.
31
Nicoletti MI, Colombo T, Rossi C, Monardo C, Stura S, Zucchetti M, et al. IDN5109, a taxane with oral bioavailability and potent antitumor activity. Cancer Res 2000;60:8426.
32
Polizzi D, Pratesi G, Monestiroli S, Tortoreto M, Zunino F, Bombardelli E, et al. Oral efficacy and bioavailability of a novel taxane. Clin Cancer Res 2000;6:20704.
33 Leonessa F, Green D, Licht T, Wright A, Wingate-Legette K, Lippman J, et al. MDA435/LCC6 WT and MDA435/LCC6mdr1: ascites models of human breast cancer. Br J Cancer 1996;73:15461.[Medline]
34 Soule HD, Vazquez J, Long A, Albert S, Brennan M. A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 1973;51:140916.[Medline]
35 Eva A, Robbins KC, Andersen PR, Srinivasan A, Tronick SR, Reddy EP, et al. Cellular genes analogous to retroviral onc genes are transcribed in human tumour cells. Nature 1982;295:1169.[Medline]
36 Rasheed S, Nelson-Rees WA, Toth EM, Arnstein P, Gardner MB. Characterization of a newly derived human sarcoma cell line (HT-1080). Cancer 1974;33:102733.[Medline]
37 Fairchild CR, Ivy SP, Kao-Shan CS, Whang-Peng J, Rosen N, Israel MA, et al. Isolation of amplified and overexpressed DNA sequences from Adriamycin-resistant human breast cancer cells. Cancer Res 1987;47:51418.[Abstract]
38 Alaoui Jamali MA, Yin MB, Mazzoni A, Bankusli I, Rustum YM. Relationship between cytotoxicity, drug accumulation, DNA damage and repair of human ovarian cancer cells treated with doxorubicin: modulation by tiapamil analog RO112933. Cancer Chemother Pharmacol 1989;25:7783.[Medline]
39 Slovak ML, Hoeltge GA, Dalton WS, Trent JM. Pharmacological and biological evidence for differing mechanisms of doxorubicin resistance in two human tumor cell lines. Cancer Res 1988;48:27937.[Abstract]
40 Lelbovitz A, Wright WC, Pathak S, Siciliano MJ, Daniels WP. Detection and analysis of a glucose 6-phosphate dehydrogenase phenotype B cell line contamination. J Natl Cancer Inst 1979;63:63545.[Medline]
41 Toffoli G, Viel A, Tumiotto L, Biscontin G, Rossi C, Boiocchi M. Pleiotropic-resistant phenotype is a multifactorial phenomenon in human colon carcinoma cell lines. Br J Cancer 1991;63:516.[Medline]
42 Turcotte JG, Srivastava SP, Steim JM, Calabresi P, Tibbetts LM, Chu MY. Cytotoxic liponucleotide analogs. II. Antitumor activity of CDPdiacylglycerol analogs containing the cytosine arabinoside moiety. Biochim Biophys Acta 1980;619:61931.[Medline]
43
Cabral F, Sobel ME, Gottesman MM. CHO mutants resistant to colchicine, colcemid or griseofulvin have an altered -tubulin. Cell 1980;20:2936.[Medline]
44 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst 1990;82:110712.[Abstract]
45 Shintani Y, Tanaka T, Nozaki Y. TI GS-164, a small synthetic compound, stimulates tubulin polymerization by a similar mechanism to that of Taxol. Cancer Chemother Pharmacol 1997;40:51320.[Medline]
46 Long BH, Carboni JM, Wasserman AJ, Cornell LA, Casazza AM, Jensen PR, et al. Eleutherobin, a novel cytotoxic agent that induces tubulin polymerization, is similar to paclitaxel (Taxol). Cancer Res 1998;58:11115.[Abstract]
47 Baily JD, Muller C, Jaffrezou JP, Demur C, Gassar G, Bordier C, et al. Lack of correlation between expression and function of P-glycoprotein in acute myeloid leukemia cell lines. Leukemia 1995;9:799807.[Medline]
48 Chaudhary PM, Roninson IB. Expression of P-glycoprotein, a multidrug efflux pump, in human hematopoietic stem cells. Cell 1991;66:8594.[Medline]
49 Drach D, Zhao S, Drach J, Mahadevia R, Gattringer C, Huber H, et al. Subpopulations of normal peripheral blood and bone marrow cells express a functional multidrug resistant phenotype. Blood 1992;80:272934.[Abstract]
50 Ludescher C, Thaler J, Drach D, Drach J, Spitaler M, Gattringer C, et al. Detection of activity of P-glycoprotein in human tumour samples using rhodamine 123. Br J Haematol 1992;82:1618.[Medline]
51 Krishan A, Ganapathi R. Laser flow cytometry and cancer chemotherapy: detection of intracellular anthracyclines by flow cytometry. J Histochem Cytochem 1979;27:16556.[Abstract]
52 Nooter K, van den Engh G, Sonneveld P. Quantitative flow cytometric determination of anthracycline content of rat bone marrow cells. Cancer Res 1983;43:512630.[Abstract]
53 Speth P, Linssen P, Boezeman JB, Wessels HM, Haanen C. Quantification of anthracyclines in human hematopoietic cell subpopulations by flow cytometry and high pressure liquid chromatography. Cytometry 1985;6:14350.[Medline]
54 Sharma A, Straubinger RM, Ojima I, Bernacki RJ. Antitumor efficacy of taxane liposomes on a human ovarian tumor xenograft in nude athymic mice. J Pharm Sci 1995;84:14004.[Medline]
55 Sharma A, Mayhew E, Bolczak L, Cavanaugh C, Harmon P, Janoff A, et al. Activity of paclitaxel liposome formulations against human ovarian tumor xenografts. Int J Cancer 1997;71:1037.[Medline]
56 Levasseur LM, Faessel H, Slocum HK, Greco WR. Implications for clinical pharmacodynamic studies of the statistical characterization of an in vitro antiproliferation assay. J Pharmacokinet Biopharm 1998;26:71733.[Medline]
57 Levasseur LM, Greco WR, Rustum YM, Slocum HK. Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells. Cancer Chemother Pharmacol 1997;40:495505.[Medline]
58 Nash JC. Compact numerical method for computers: linear algebra and function minimization. New York (NY): John Wiley & Sons; 1979.
59 Peto R, Peto J. Asymptotically efficient rank invariant test procedures. J R Stat Soc 1972;135A:185206.
60 Alvarez M, Paull K, Monks A, Hose C, Lee JS, Weinstein J, et al. Generation of a drug resistance profile by quantitation of mdr-1/P-glycoprotein in the cell lines of the National Cancer Institute Anticancer Drug Screen. J Clin Invest 1995;95:220514.[Medline]
61 Frankel A, Buckman R, Kerbel RS. Abrogation of Taxol-induced G2-M arrest and apoptosis in human ovarian cancer cells grown as multicellular tumor spheroids. Cancer Res 1997;57:238893.[Abstract]
Manuscript received July 28, 2000; revised May 22, 2001; accepted June 13, 2001.
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