THYROID Volume 12, Number 12, 2002 © Mary Ann Liebert, Inc.
Combretastatin A4 Phosphate Has Primary Antineoplastic Activity Against Human Anaplastic Thyroid Carcinoma Cell Lines and Xenograft Tumors Joshua M. Dziba, Regina Marcinek, Gopalakrishnan Venkataraman, Jill A. Robinson, and Kenneth B. Ain
Anaplastic thyroid carcinoma (ATC) is a fatal malignancy the clinical outcome of which is unaltered by current therapeutic modalities. A recent phase 1 clinical trial of combretastatin A4 phosphate (CA4P) produced a long-lasting total remission in a patient with ATC. CA4P is a tubulin-binding agent derived from the African bush willow, Combretum caffrum, which possesses tumor vascular-targeting activity. In order to discriminate primary antineoplastic effects from tumor antivascular activity, we evaluated CA4P cytotoxicity in eight human ATC cell lines and compared it to paclitaxel, another tubulin-binding agent with significant clinical activity. CA4P displayed significant cytotoxicity against the ATC cell lines, comparable to that of paclitaxel, and these effects were longer lasting in two cell lines compared to the duration of paclitaxel. We further investigated the effects of CA4P on xenograft tumors from four ATC cell lines injected in athymic nude mice. Significantly lower tumor weights were observed in animals treated with CA4P compared to those treated with vehicle alone. Continuous monitoring of xenograft tumor volumes from one of the ATC cell lines also revealed a significantly lower rate of tumor growth in the CA4P-treated mice compared to those receiving vehicle alone. These results suggest that antitumoral effects of CA4P can be consequent to a combination of primary antineoplastic effects as well as the potential destruction of tumor vasculature.
Introduction
A
(ATC) is a rare, but extremely aggressive type of cancer that is essentially always fatal, usually within 6 months of diagnosis (1). Recently, we showed that paclitaxel, a drug derived from the Pacific yew tree (Taxus brevifolia), had significant cytotoxic activity against ATC (2,3). Despite this, ultimate disease-specific mortality is unaltered, demonstrating the need for additional effective therapies. The requirement for neovascularization to support progression of tumors has inspired a new target for therapeutic efforts (4). Investigations have shown that various tumor vascular-targeting drugs that destroy the vasculature of developing tumors can arrest, and even reverse tumor progression (5–7). Combretastatins, a class of drugs isolated from the bark of the African bush willow tree, Combretum caffrum, particularly the prodrug derivative, combretastatin A4-phosphate (CA4P), have been evaluated as vasculartargeting agents with significant preclinical activity (6,8,9). CA4P is of particular interest as a possible treatment for ATC NAPLASTIC THYROID CARCINOMA
because one patient with ATC is presumed to have experienced a durable complete response in a phase 1 trial as a result of treatment with the drug (10). Although the mechanism of activity of CA4P may be because of vascular-targeting properties, there is a possibility that the antitumoral effects also reflect the contribution of direct cytotoxicity. While others have shown that CA4P possesses antivascular effects against proliferating, nonneoplastic tissue (11), CA4P has also been shown to possess direct antineoplastic activity against different tumor cell lines (12). Other derivatives of combretastatin, as well as the most potent member, CA4P, have also been shown to possess direct antineoplastic properties (13–16). In order to evaluate the potential role of CA4P as a component of the therapy of ATC, we undertook an in vitro analysis of its cytotoxic activity using human ATC cell lines. We investigated the antineoplastic properties of CA4P against eight human anaplastic thyroid cancer cell lines (ARO81, BHT101, C643, DRO-90, KAT-4, SW1736, HTh7, and HTh74), comparing the cytotoxic effects of CA4P to paclitaxel, both alone as well as in combination. In four of these cell lines,
Thyroid Cancer Research Laboratory, Medical Service, Veterans Affairs Medical Center, and the Department of Internal Medicine, University of Kentucky Medical Center, Lexington, Kentucky.
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DZIBA ET AL.
grown as xenograft tumors in nude mice, in vivo activity was also studied. Materials and Methods
Cell lines Cell lines were derived from primary cultures of human anaplastic thyroid carcinoma tumors. DRO-90 (DRO) and ARO81 (ARO) were provided by Dr. G.F.J. Juillard (Department of Radiation Oncology, University of California-Los Angeles School of Medicine). BHT-101 (BHT) was provided by Dr. I. Pályi (National Institute of Oncology, Budapest, Hungary) (17). SW1736 was developed by Leibowitz and McCombs III at the Scott and White Memorial Hospital (Temple, TX) in 1977 and was provided by Dr. Nils-Erik Heldin (Uppsala University, Uppsala, Sweden). C643, HTh7, and HTh-74 were also provided by Dr. Heldin (18). KAT4 was developed and is maintained by our laboratory.
Mice Athymic (nu/nu) nude male mice (NCI, Frederick, Maryland) were housed in cages in groups of four animals each, and were fed ad libitum. Mice were injected subcutaneously with ATC cell lines at approximately 7 weeks of age.
Reagents Combretastatin A-4 phosphate di-sodium salt (CA4P) (19) was provided by Dr. Ravi Varma, Drug Synthesis and Chemistry Branch, National Cancer Institute, National Institutes of Health (NSC 643812-F/3) and Oxigene, Inc. (Watertown, MA). CA4P was dissolved in 0.9% sodium chloride (NaCl). Paclitaxel (Taxol® , BMS-181339-01) was provided by BristolMyers Squibb Co. (Princeton, NJ) and was dissolved in dimethylsulfoxide (DMSO). All analytical grade reagents were purchased from Sigma Chemical Co. (St. Louis, MO) with the exception of fetal bovine serum (FBS), phosphatebuffered saline (PBS), Hank’s buffered salt solution (HBSS), RPMI 1640 medium (RPMI), nonessential amino acids, sodium pyruvate (NaPyr), and trypsin-ethylenediaminetetraacetic acid (EDTA) which were purchased from Gibco/ Life Technologies (Gaithersburg, MD).
Cell culture conditions Cultured cells were maintained in 75-cm2 culture flasks (Sarstedt, Newton, NC), in an atmosphere of 5% CO2, 95% humidity, and at 37°C. All cell lines were thawed from stocks maintained in liquid nitrogen into RPMI media supplemented with 10% FBS, 0.1 mM nonessential amino acids, and 1.0 mM NaPyr. The DMSO-containing freezing media was replaced with fresh media soon after the cells attached to the surface of the flasks. Cells were seeded to either 12-well or 24-well culture plates (Becton Dickinson Labware, Franklin Lakes, NJ, and Corning Inc., Corning, NY, respectively). The cytotoxicity assays were conducted in media with 10% FBS while the recovery experiments were conducted in media with 5% FBS.
Dose-response experiments Each of the cell lines was tested for response to a range of doses of CA4P or paclitaxel. Cells were at 5%–10% confluence when the drugs were first added. All cell number quantifications were performed on nonconfluent cultures. At 48 hours, cells were treated with different doses of either CA4P, dissolved in 0.9% NaCl, or paclitaxel in DMSO. Each dose administration was evaluated in six replicate wells with entire experiments repeated in duplicate. Control cells received an equal volume of the corresponding diluent alone. Viable cell quantification was represented by the microculture 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay (20,21) with baseline values obtained just prior to adding test drugs (20,21). Cells were exposed to drugs for 72 hours prior to cell quantification.
Combination of CA4P with paclitaxel Cells were seeded, as above, and after 48 hours the cells were treated with their respective GI50 concentrations of CA4P (as determined from the dose-response experiments) in combination with a wide range of paclitaxel dosages. In each experiment, the constant GI50 reagent was added immediately prior to the varying dose reagent. The GI50 value is measured as the concentration of an agent at which 50% growth inhibition of the cells is observed (22). Alternatively,
TABLE 1. ANTINEOPLA STIC PARAMETERS DETERMINED
FROM
DOSE -RESPONSE EXPERIMEN TS
Parameter CA4P (nM) ATC cell line ARO81 BHT-101 HTh7 DRO-90 C643 KAT-4 SW1736 HTh74
GI50 2.3 3.5 2.5 3.5 4.5 . 227.5 3.5 3.5
TGI
. . . . .
3.5 8.5 9.5 227.5 227.5 227.5 227.5 227.5
Paclitaxel (nM) LC50
. . . . . .
71.8 20.5 227.5 227.5 227.5 227.5 227.5 227.5
GI50
TGI
LC50
1.5 30.5 2.8 1.7 5.5 6.5 1.6 1.8
3.8 . 31.6 15.5 4.5 . 31.6 . 31.6 3.5 9.5
20.5 . 31.6 . 31.6 10.5 . 31.6 . 31.6 15.5 . 31.6
Cells were treated with either CA4P or paclitaxel for 72 hours prior to measurement of cell viability. The concentrations resulting in a 50% inhibition of growth (GI50 ), a total inhibition of cellular growth (TGI), or lethality to 50% of the cells present prior to drug administration (LC50 ) were determined.
CA4P AND ANAPLASTIC THYROID CARCINOMA
1065 was injected intraperitoneally every day for two 5-day periods separated by 2 days without injections. In a separate experiment (using BHT-101 xenografts), 10 doses were administered on alternate days. Subcutaneous tumors were measured at the same time with calipers, and tumor volumes were calculated by the formula; a2 3 b 3 0.4, where a is the shortest diameter and b is the diameter perpendicular to a (23). Mice were killed 2 days after the administration of the final CA4P dose. Tumor measurements, including final tumor weights, were recorded on the day the animals were killed.
Data analysis
FIG. 1. Dose-dependent growth of anaplastic thyroid carcinoma (ATC) cell lines in CA4P. Cells were treated with CA4P for 72 hours prior to measurement of cell viability. Each point is the mean (6 standard error of the mean [SEM]) of the 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) activity of six culture wells. MTT activity is expressed as a percentage relative to the untreated control cells.
Antineoplastic growth parameters, TGI, GI50, and LC50 (total growth inhibition, 50% growth inhibition, and concentration resulting in 50% lethality, respectively), were evaluated for each of the cell lines (Table 1) (24). The TGI value is the concentration of drug at which a complete cytostatic effect is observed. The LC 50 is the concentration of drug that causes a 50% reduction in the number of cells, compared to the number of cells initially present at the beginning of the experiment. In the dose-response experiments, the growth response of the cells treated with drugs was normalized to the control groups, exposed to the diluents alone. A relative MTT activity of 100 indicates no drug effect, whereas MTT activity values approaching zero indicate increasing cytostatic drug effects. Relative MTT activity below zero indicates increasing cytotoxicity. In the growth recovery exper-
cells were treated with the respective GI50 concentrations of paclitaxel in combination with a wide range of CA4P. Control cells received equal volumes of respective diluents in lieu of test drugs. Cells were exposed to the test drugs for 72 hours prior to quantifying viable cells by MTT.
Cell growth recovery experiments Cells were seeded in 12-well culture dishes at concentrations ranging from 1.5 3 104 to 2.5 3 104 cells per well with four replicate wells for each condition. After 48 hours, cells were treated with either CA4P or paclitaxel. Control cells received equal volumes of the respective diluent. Viable cells were quantified using the MTT assay in parallel cultures, just prior to adding the drugs. Cells were treated for 72 hours, followed by aspiration of medium, then rinsed with HBSS and replacement of fresh culture medium without drug. Cells were quantified at this point and then at 24-hour intervals, in parallel cultures, for 5 consecutive days after removal of the test drugs.
Xenograft studies Four cell lines (ARO81, BHT-101, DRO-90, and KAT-4) were individually injected subcutaneously in a flank of each of 24 athymic nude male mice, as suspensions of 2 3 106 cells in PBS. Treatment and control groups consisted of 6 mice each for each of the cell lines. When xenograft tumors reached volumes of 200–300 mm3, the mice were distributed between treatment and control groups to normalize the mean tumor volumes prior to administration of CA4P. Control mice received equal volumes of the saline diluent. One hundred fifty milligrams per kilogram of CA4P (or diluent alone)
FIG. 2. Dose dependent growth of anaplastic thyroid carcinoma (ATC) cell lines in paclitaxel. Cells were treated with paclitaxel for 72 hours prior to measurement of cell viability. Each point is the mean (6 standard error of the mean [SEM]) of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) activity of six culture wells. MTT activity is expressed as a percentage relative to the untreated control cells.
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DZIBA ET AL. over a 72-hour period, caused varied cytotoxicity in the eight cell lines (Table 1). The antineoplastic parameters for CA4P and paclitaxel were used to evaluate the cytotoxicity of each drug in each of the eight cultures. GI50 values for CA4P were remarkably similar in all but the KAT-4 cell line, which was much more resistant to the antineoplastic effects of CA4P, with greater differences exhibited for the TGI and the LC 50 results between cell lines. TGI was observed in three cell lines with CA4P concentrations under 10 nM (ARO81, BHT-101, and HTh7); however, paclitaxel completely inhibited cell growth in five cell lines (ARO81, DRO-90, HTh7, HTh74, and SW1736) at similar concentrations. The ARO81 and BHT-101 cell lines were sufficiently sensitive to the cytotoxic effects of CA4P to enable calculation of LC50 values within the concentrations studied. LC 50 values for ARO81 and BHT-101 were 71.77 nM and 20 nM, respectively. LC 50 values for the other six cell lines exceeded the CA4P drug concentrations used. TGI values for the ARO81, BHT-101, and HTh7 cells were: 3.5 nM, 8 nM, and
FIG. 3. Growth of anaplastic thyroid carcinoma (ATC) cell lines exposed to a constant dose of paclitaxel and a range of doses of CA4P. Cells were plated and cultured for 48 hours, then treated with the concentration of paclitaxel representing the 50% growth inhibition (GI50) dose (respectively, for each cell line) in combination with a range of CA4P dosages. Cells were treated with the combination for 72 hours prior to measurement of cell viability. Each point is the mean (6 standard error of the mean [SEM]) of the MTT activity of six culture wells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) activity expressed as a percentage relative to the untreated control cells. The wide-dashed line represents the relative MTT activity of cells treated with the GI50 paclitaxel dose without any CA4P. Analysis of variance (ANOVA) with repeated measures (p , 0.0001).
iments, the growth rates were represented as the slopes of the cell culture growth over the recovery time (after removing active drug), with the relative growth rates expressed as a percentage of the corresponding control growth rates. Statistical analysis of data was done using the program, StatView 5 for the Macintosh (SAS Institute Inc., Cary, NC). Monolayer studies were analyzed using Dunnett’s test for multiple comparisons (25,26). Xenograft tumor-volume data were evaluated using analysis of variance (ANOVA) with repeated measures while tumor weights were analyzed using the unpaired t test. Results
Effect of CA4P Exposure of ATC cultures to various concentrations of CA4P concentrations, ranging from 0.717 nM to 227.1 nM
FIG. 4. Growth of anaplastic thyroid carcinoma (ATC) cell lines exposed to a constant dose of CA4P and a range of doses of paclitaxel. Cells were plated and cultured for 48 hours, then treated with the concentration of CA4P representing the GI50 dose (respectively, for each cell line) in combination with a range of paclitaxel dosages. Cells were treated with the combination for 72 hours prior to measurement of cell viability. Each point is the mean (6 standard error of the mean [SEM]) of the 3-(4,5-dimethylthiazol-2-yl)2,5,-diphenyl-tetrazolium bromide (MTT) activity of six culture wells. MTT activity is expressed as a percentage relative to the untreated control cells. The wide-dashed line represents the relative MTT activity of cells treated with the same CA4P dose used, without paclitaxel. Analysis of variance (ANOVA) with repeated measures (p , 0.0001).
CA4P AND ANAPLASTIC THYROID CARCINOMA
FIG. 5. Growth recovery curves for anaplastic thyroid carcinoma (ATC) cell lines exposed to CA4P. Cells were cultured for 48 hours, treated with CA4P for 72 hours, and then replenished with fresh culture medium with no drug. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) activity, expressed as a percentage relative to the untreated control cells, was measured at this time and again, at 24-hour intervals, for 5 consecutive days. Each point is the mean (6 standard error of the mean [SEM]) of four culture wells.
9.5 nM, respectively; corresponding to concentrations at least one order of magnitude less than found effective in the remaining four ATC cell lines. The KAT-4 cell line was least responsive to the exposure to CA4P, while the ARO81 and BHT-101 cell lines were most responsive (Fig. 1).
Effects of CA4P in combination with paclitaxel Paclitaxel, as a single agent, revealed distinct patterns of activity in the eight ATC cell lines (Table 1) in cell culture. Exposure of ARO81, DRO-90, SW1736, HTh7 and HTh74 cells to paclitaxel concentrations of 3 nM or more, for more than 72 hours, caused significant cytotoxicity while TGI values for the other seven cell lines were above the concentrations studied (. 31.6 nM). The BHT-101 cell line was least responsive to paclitaxel, while the DRO-90 cells were most responsive (Fig. 2). Combination treatment with paclitaxel and CA4P was evaluated in three cell lines (BHT-101, DRO-90, and HTh7). In separate experiments, single concentrations of paclitaxel, equivalent to the GI50 concentration for each respective cell line, were added to a spectrum of CA4P concentrations. In additional experiments, single concentrations of CA4P, likewise corresponding to GI50 values in the respective cell lines, were used with a spectrum of paclitaxel doses. In each set of experiments, the constant dosed agent was added prior to adding the varying dosed agent. In Figures 3 and 4, the wide dashed lines indicate the relative MTT activity of cells treated with the GI50 concentration of the constant dosed agent alone. Studies with BHT-101 and HTh7 in which the concentration of paclitaxel was held constant while varying the concentration of CA4P revealed cytotoxicity levels at the lower concentrations of CA4P, similar to the cytotoxicity
1067 level of paclitaxel alone (Fig. 3). At the higher concentrations of CA4P (at or above 7.18 nM) the combination of the two drugs was just as lethal as the CA4P alone. This suggests the lack of antineoplastic interaction, neither additive nor antagonistic, because of the combination. Studies in which the concentration of CA4P was held constant while varying the concentration of paclitaxel revealed some additive cytotoxicity due to the combination in the three cell lines (Fig. 4). In the BHT-101 cells, additive effects were observed at paclitaxel concentrations higher than 1 nM, and extended up to the highest concentration tested, 31.6 nM. In the HTh7 cells, additive antineoplastic effects were observed at paclitaxel concentrations higher than 1 nM but appeared to dissipate beyond 31.6 nM. In studies with DRO90 cells, the combination of low doses of paclitaxel (0.1 to 2.0 nM) and constant CA4P was less cytotoxic than CA4P alone. There was no enhanced cytotoxicity over paclitaxel alone, from the combined use of constant dose CA4P when paclitaxel doses were at least 10 nM.
Posttreatment recovery Four cell lines (ARO81, BHT-101, DRO-90, and HTh7) that were responsive to CA4P in the dose-response experiments (Table 1) were further studied for their ability to recover and grow after being exposed to CA4P or to paclitaxel in monolayer cultures. None of these four cell lines were able to regain their baseline rate of growth within the defined observation period after removal of the respective TGI levels of CA4P. ARO81 and BHT-101 cells treated with the GI50 concentration of CA4P recovered at a 10-fold lower proliferation rate than the corresponding controls (Table 2). BHT-101 cells did not grow significantly after removal of the GI50 concentration of CA4P (Fig. 5). DRO-90 cells demonstrated the most dramatic recovery of growth after removal of the GI50 concentration of CA4P, with a rate only 5% less than the controls (Table 2). HTh7 cells displayed partial recovery, with a rate one third of the control rate. ARO81 cells treated with paclitaxel did not effectively recover, showing a relative growth rate of 0.04, while the BHT-101 and DRO-90 cells had relative growth rates comparable to controls at more than 0.70 and 0.90, respectively (Fig. 6, Table 2). HTh7 cells showed partial recovery with a rate of about 0.44. TABLE 2. RELATIVE GROWTH RECOVERY FOR ATC MONOLAYER CULTURES TREATED WITH EITHER CA4P OR PALITAXEL Relative growth recovery after drug removal (Ratio of growth rate:treated cells vs. untreated control cells) Cell line
CA4P
Paclitaxel
ARO81 BHT-101 DR0-90 HTh 7
0.084 0.120 0.954 0.376
0.040 0.733 0.903 0.439
Cells were cultured for 48 hours, treated with either CA4P or paclitaxel or the corresponding diluent alone, for 72 hours and then replenished with fresh culture medium with no drug. The ratio for each condition is obtained by dividing the slope of a linear fit for the recovery growth curve of the drug treated cells with the corresponding slope of the control cells. A ratio of 1.0 denotes a growth rate equal to the corresponding control cell cultures.
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FIG. 6. Growth recovery curves for anaplastic thyroid carcinoma (ATC) cell lines exposed to paclitaxel. Cells were cultured for 48 hours, treated with paclitaxel for 72 hours, and then replenished with fresh culture medium with no drug. The 3-(4,5-dimethylthiazol-2-yl)-2,5,-diphenyl-tetrazolium bromide (MTT) activity, expressed as a percentage relative to the untreated control cells, was measured at this time and again, at 24-hour intervals, for 5 consecutive days. Each point is the mean (6 standard error of the mean [SEM]) of four culture wells.
Effects of CA4P on xenograft tumors The growth of ATC cells as xenograft tumors was monitored during the course of administration of 150 mg/kg CA4P or saline diluent to the mice. Subsequent tumor volumes were normalized to the volumes at the point of administration of the first dose of either the drug or vehicle. Administration of CA4P for 5 continuous days resulted in
FIG. 7. Final xenograft tumor weights: CA4P vs. saline. CA4P (150 mg/kg) or saline vehicle was administered intraperitoneally daily for two periods of 5 days each, interrupted by a 2-day rest period. Tumors were excised 2 days after the last dose and weighed. Only xenografts that completed the CA4P treatment course were measured (ARO, BHT, DRO, KAT-4, and controls; n 5 4, 2, 4, 5, and 6 mice, respectively) CA4P-treated tumors were had significantly reduced weights compared to control tumors (*p , 0.05). Bars represent means for each group (6 standard error of the mean [SEM]).
DZIBA ET AL. the death of 9 of 24 mice that had received the drug, compared to no fatalities in the groups of animals receiving saline. Analysis of tumor weights from the mice that survived the full course of drug administration indicated significantly lower tumor weights from CA4P treated mice compared to saline-treated mice in the ARO and DRO xenograft mice groups (Fig. 7). Lower tumor volumes were also noted in the BHT and KAT-4 mice groups, although they did not reach statistical significance. Regular administration of 10 doses of 150 mg/kg CA4P on alternate days resulted in the death of one BHT xenograft mouse that had received CA4P, and no fatalities of mice receiving saline alone. Tumor growth rates of the BHT xenografts were markedly less in mice receiving alternate doses of CA4P compared to the saline-treated mice (Fig. 8) with lower final tumor weights also noted compared to those of tumors from saline-treated mice. The tumor in 1 of the 6 mice receiving CA4P on alternate days completely disappeared by the 18th day of drug administration. The tumor, although not measurable with calipers, was palpable. One mouse died on the 11th day of the drug administration period, just before administration of the 6th dose of CA4P. Discussion ATC remains incurable despite the use of paclitaxel or any other standard systemic antineoplastic agents. More potent chemotherapeutic agents that can be used alone or in combination with existing therapies could have a major impact on disease prognosis. CA4P has been investigated extensively for its angiolytic properties in malignant tumors with one study indicating possible clinical effectiveness, which may be enhanced by concomitant antineoplastic activity (10). Other studies have shown that CA4P, as well as some CA4P
FIG. 8. BHT-101 xenograft tumors treated with CA4P or vehicle. CA4P (150 mg/kg) or saline vehicle was administered intraperitoneally every 48 hours for 10 doses and tumors measured with calipers. The graph shows means for tumor volumes normalized to the initial volumes prior to administration of either CA4P (n 5 6 before day 12, and n 5 5 from day 12) or saline (n 5 6) (p , 0.01). By the end of the experiment, one of the CA4P-treated mice had no evidence of their xenograft (*).
CA4P AND ANAPLASTIC THYROID CARCINOMA analogues, have direct cytotoxic effects against neoplasms. Our analysis of the cytotoxic effects of CA4P against ATC, as a single agent or in combination with paclitaxel, revealed significant antineoplastic responses that were equal or greater than those seen with paclitaxel. This suggests that CA4P may be an active antineoplastic agent for human ATC, independent of any vascular-targeting properties. Studies using the combination of CA4P and paclitaxel utilized constant concentrations of one agent, corresponding to the individual GI50 concentrations of that drug, with a wide range of dosages of the second agent. In this way, synergistic or antagonistic effects could be revealed. The BHT-101 and HTh7 cells were the only cell lines that exhibited enhanced cytotoxicity in response to a combination of CA4P with paclitaxel. Such synergistic effects were only seen in experiments in which a constant concentration of CA4P was combined with varying concentrations of paclitaxel. The interaction of CA4P and paclitaxel is uncertain because they both bind tubulin with a similar tubulin-binding site; however, paclitaxel stabilizes tubulin polymerization while CA4P can displace radiolabeledcolchicine from tubulin and inhibits tubulin polymerization (27,28). This uncertain interaction may suggest the consideration of other antineoplastic agents, in combination with CA4P, which utilize completely different mechanisms with greater chance for true antineoplastic synergy. The absence of a consistent difference in growth rate recovery after exposure of ATC cell lines to CA4P, compared to paclitaxel, suggests the application of similar dosing frequencies. On the other hand, unique cell lines or unique patient tumors may benefit by a prolonged clinical response to CA4P using infrequent dosing intervals (Table 2). Specific pharmacokinetic studies in humans must also account for the confounding effect of antiangiogenic activity of CA4P resulting in a diminished direct cytotoxic effect by reducing CA4P accessibility to the tumor. Likewise, the translation of in vitro CA4P concentrations to in vivo CA4P plasma concentrations is problematic at best. Responsive ATC cell lines demonstrated maximal antineoplastic response at CA4P concentrations exceeding 0.01 mM. The administration of 100 mg/kg CA4P to a mouse produces a plasma CA4P level of just under 3.0 mM (29), roughly estimated to correspond to a human dose of 0.08 mg/kg (30). Recent pharmacokinetic studies suggest that the maximal tolerated human dose is around 60 mg/m2 or 1.6 mg/kg (assuming a 70-kg person) (10). Thus, even accounting for a high level of protein binding, it is likely that antineoplastic CA4P concentrations would be tolerated in human administration. In each of the in vivo experiments using CA4P against ATC xenografts, significant antitumor activity was evident. We cannot yet discern whether this represents cytotoxicity, vascular targeting, or a combination of these effects. Further analysis quantifying tumor vascularity between control and treatment groups may offer some answers. Because vascular-targeting activity often produces cavitation of tumors (31,32) with a viable peripheral tumor rim, combinations of CA4P with antineoplastic agents may prove most active. Acknowledgment We are indebted to Dr. G.R. Pettit (Arizona State University, Tempe, AZ) for providing research reagents, stimulating ideas, and useful advice in the course of these studies.
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