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Original Studies |
Section of Endocrine Neoplasia and Hormonal Disorders, Department of Internal Medicine Specialties (G.X., J.P., S.-C.J.Y.), and Section of General Internal Medicine, Department of Internal Medicine Specialties (C.M., S.-C.J.Y.), University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030
Address all correspondence and requests for reprints to: S. Jim Yeung, M.D., Ph.D., University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 40, Houston, Texas 77030. E-mail: syeung{at}notes.mdacc.tmc.edu
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Abstract |
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Introduction |
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A new group of therapeutic agents called farnesyltransferase inhibitors (FTIs) is now under investigation for use in the treatment of solid tumors. FTIs may be useful against thyroid cancers, because activating ras mutations and ret tyrosine kinase activated by rearrangement (PTC oncogenes) are common in thyroid cancers (9). Ras, the protein product of ras protooncogenes, is synthesized as a cytosolic precursor, and it requires posttranslational modification by conjugation of a farnesyl (15-carbon isoprenyl group) moiety to the C-terminal. After farnesylation, Ras is localized to the inner surface of the cell membrane and becomes functional in transducing the mitogenic signals of tyrosine kinase receptors. The antineoplastic activity of FTIs results at least in part from inhibiting Ras and thus blocking the mitogenic signaling pathway of tyrosine kinase growth receptors. Recently, other farnesylated proteins, such as RhoB (10, 11), have been demonstrated to play a role in the antineoplastic activity of FTIs. Manumycin A, a natural product of Streptomyces parvulus, inhibits farnesyltransferase by competing with the farnesyl pyrophosphate substrate, and it has antitumor activity in vitro and in xenograft models against a variety of cancers (12, 13, 14, 15).
Our previous works have demonstrated that the combination of paclitaxel and manumycin improved their antineoplastic effect against ATC cells (15). In this paper we present data further showing that the combination of paclitaxel and manumycin provides improved antineoplastic activity in vivo without increased toxicity. We observed that the tumor xenografts that were treated with manumycin were paler than those not exposed to manumycin. Because the pale color suggested decreased vascularity, and angiogenesis is an important factor regulating tumor growth in vivo (16), we investigated whether the antineoplastic effect of manumycin and paclitaxel against ATC cells in vivo was at least in part mediated by inhibition of tumor angiogenesis. Angiogenesis is a continuous process, as is tumor growth. We also hypothesized that sustained delivery of manumycin would improve antineoplastic activity over intermittent injection of manumycin. Therefore, we compared the efficacy of the combination of paclitaxel with slow release manumycin to that of the combination of paclitaxel with ip manumycin against ATC xenografts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Seven-week-old nude mice (nu/nu BALB-c mice bred at the animal facility of University of Texas M. D. Anderson Cancer Center) were used in this study. The mice were housed in barrier facilities on a 12-h light/dark cycle with food and water available ad libitum. The animals were cared for according to the NIH Guide for the Care and Use of Laboratory Animals.
Drugs
Manumycin A and paclitaxel were purchased from Sigma (St. Louis, MO). They were dissolved in dimethylsulfoxide (DMSO; Sigma) at appropriate concentrations before dilution in tissue culture medium such that the final concentration of DMSO would not exceed 0.1% (vol/vol).
Cell culture
Four human ATC cell lines were used: ARO (4), KAT-4 (3), KAT-18 (3), and Hth-74 (17). The cells were cultured as previously described (15). Human umbilical vein endothelial cells (HUVECp), human microvascular endothelial cells (HMVECad), specialized media, growth factors, and attachment factors were obtained from Cascade Biologicals, Inc. (Portland, OR), and cultured according to the protocols provided by the supplier. Hypoxic culture conditions were induced using BBL GasPak Plus (Becton Dickinson and Co., Cockeysville, MD) and were confirmed using dry anaerobic indicator strips (Becton Dickinson and Co.).
Nude mouse xenograft model
Human ATC cells (1 x 106) suspended in RPMI 1640 medium were injected sc into a flank of each 7-week-old nude mouse (nu/nu BALB-c, bred at our institution). Tumor volumes were calculated using the formula: a2 x b x 0.4, in which a is the smallest diameter, and b is the diameter perpendicular to a. After the tumors reached at least 10 mm3, the mice were randomly assigned to groups. Drug solutions (manumycin, 7.5 mg/kg·dose, paclitaxel, 20 mg/kg·dose) or placebo (0.1% DMSO in tissue culture medium) were injected ip on days 1 and 3 of a 7-day cycle for three cycles. Manumycin slow release pellets (1.25 mg released over 30 days; custom produced by Innovative Research of America, Toledo, OH) or placebo pellets were implanted sc using a trochar at about 1 cm from the puncture sites. There were four animals per treatment group. Serial measurements of tumor sizes were plotted as growth curves [i.e. log (tumor volume/original volume) vs. days since treatment began] with the error bars representing the SEs.
The doses of manumycin (15 mg/kg·week) and paclitaxel (40 mg/kg·week) chosen were about half of the high doses reported in the literature [i.e. manumycin, 31.25 mg/kg·week (13); paclitaxel, 80 mg/kg·week (3)]. Based on the average body weight of a 7-week-old nude mouse being about 22 g, the amount of manumycin in a 30-day sc slow release tablet was chosen to deliver 13.25 mg/kg·week, which was slightly lower than the dose of ip manumycin.
The body weight, feeding behavior, and motor activity of each animal were monitored as indicators of general health. Food intake was partially reflected by the body weight, and feeding behavior (the ability to feed) was observed upon presentation of food. Motor activity was assessed by avoidance behavior and the ability to escape when the observer tried to capture the animal. When the animals were killed, blood samples were collected by intracardiac puncture, and tumor samples were dissected. Lactate dehydrogenase, aspartate and alanine transaminases, and alkaline phosphatase levels were measured, and complete blood count with differential were obtained in the veterinary clinical laboratory at our institution. Half of each tumor was fixed in 10% neutral buffered formaldehyde (Sigma), and the other half was snap-frozen and stored in liquid nitrogen.
Angiogenesis in Matrigel implants
Matrigel implants in mice have often been used as an in vivo assay for angiogenesis (18, 19, 20). The degree of angiogenesis was measured histologically (18) and by assaying for hemoglobin (19) in the Matrigel (Collaborative Biomedical Products, Bedford, MA) implants. We injected 0.5 mL Matrigel containing 60 ng vascular endothelial growth factor (VEGF) and 10 ng basic fibroblast growth factor (bFGF) (19) sc in 6-week-old nude mice. Each mouse received two Matrigel implants. The mice were randomized into four groups [control (placebo), manumycin, paclitaxel, and manumycin plus paclitaxel] and received ip drug injections [manumycin (7.5 mg/kg·dose), paclitaxel (20 mg/kg·dose), or placebo] on days 1 and 3 of a 7-day cycle for two cycles. Then the animals were killed, and the Matrigel implants were harvested. For each animal, we measured the hemoglobin concentration in one implant using the Drabkin method (Sigma). The surrounding adherent tissue was carefully dissected away from each harvested Matrigel implant. The implant was melted on ice and mixed with Drabkin’s reagent. The optical density (OD) at 545 nm was measured using SpectraFluor Plus (Tecan, Inc., Research Triangle Park, NC). For the histomorphometric analysis, the other implants from each animal were fixed in 10% neutral buffered formaldehyde solution (Sigma), embedded in paraffin, and cut into 4-µm sections. Half of the slides were stained with Masson’s Trichrome stain, and the vessel area and total Matrigel area in digitized photomicrographs were measured in pixels using a version of NIH Image. The other half of the slides was stained with hematoxylin-eosin. Random microscopic fields were photographed and digitized, and the nuclei were identified and counted.
SDS-PAGE and immunoblotting
SDS-PAGE was performed with standard methods. Western blotting was performed using polyvinylidine difluoride membranes and chemiluminescence. The primary antibody was polyclonal rabbit anti-VEGF antibody (Calbiochem, San Diego, CA), and the secondary antibody was antirabbit IgG-peroxidase conjugate (Roche Molecular Biochemicals, Indianapolis, IN).
Immunohistochemistry
Immunostaining was performed on ATC xenograft sections using the avidin-biotin peroxidase complex method (Vector Laboratories, Inc., Burlingame, CA) according to standard procedures. The primary antibody was a rabbit polyclonal antibody of VEGF (Calbiochem) at a 1:50 dilution. The secondary antibody was biotinylated antirabbit IgG (Vector Laboratories, Inc.) at a 1:200 dilution. Color was developed with 3,3'-diaminobenzidene tetrahydrochloride, and the slides were counterstained with hematoxylin. A negative control was included for each xenograft specimen by substituting the primary antibody with normal goat serum.
Wst-1 cell proliferation assay
The number of viable cells was measured using the Cell Proliferation Kit II (WST-1, Roche Molecular Biochemicals) according to standard protocols. The cells were cultured in 96-well plates except in the transmembrane migration assay. The amount of formazan product, measured as the OD at wavelength 450 nm (OD 450) with reference at wavelength 620 nm (OD 620), directly correlates with the number of metabolically active cells. As a negative control for background OD, 70% alcohol was used to kill the cells. All experiments were performed in quadruplicate and with the OD values inside the linear range. The mean OD values were plotted with the error bars representing the 95% confidence intervals.
[3H]Thymidine incorporation
Cell proliferation was assessed by the incorporation of radiolabeled thymidine into trichloroacetic acid (TCA)-precipitable material. HUVECp were plated in 96-well culture plates at densities of 1.0 x 104 cells/well and labeled with [methyl-3H]thymidine at 1 µCi/mL for the last 2 h of a 48-h incubation in different concentration of drugs. After labeling and experimental treatments, the cells were detached by trypsin-ethylenediamine tetraacetate treatment and transferred to a well in the Bio-Dot apparatus (Bio-Rad Laboratories, Inc.) with 0.5-mm-thick chromatography paper (Whatman, Newton, MA). The cells were filtered using suction onto the paper and were washed twice with 5% TCA and twice with 95% ethanol. The area of dried paper corresponding to each well was cut out with a round hole-punch, and the radioactivity was counted by liquid scintillation using a Tri-Carb 2100TR analyzer (Packard Instruments, Meriden, CT). The experiment was performed in quadruplicate, and the mean TCA-precipitable radioactivity values were plotted with the error bars representing the 95% confidence intervals.
Transmembrane migration assay
Migration of endothelial cells was tested with the FluoroBloc Transwell tissue culture inserts (Becton Dickinson and Co.) with 8-µm pores. The inserts were placed into a well of a 24-well plate containing 600 µL medium supplemented with 60 ng VEGF and 10 ng bFGF in the presence or absence of manumycin and/or paclitaxel. The inner chamber of each FluoroBloc contained 400 µL regular medium with endothelial cells that had been labeled with DiO fluorescent dye (Molecular Probes, Inc., Eugene, OR) by incubation overnight. After 4, 8, and 24 h of incubation, fluorescence at the bottom surface of the FluoroBloc was measured using SpectraFluor Plus (Tecan, Inc.), with excitation wavelength of 485 nm and emission wavelength of 520 nm. At the end of the experiment, 40 µL WST-1 dye (Roche Molecular Biochemicals) were added to the inner chamber and incubated at 37 C for an additional hour to measure the viability of the cells. The experiment was performed in triplicate, and the mean fluorescence values were plotted with the error bars representing the 95% confidence intervals.
Tube formation assay
Matrigel has been frequently used in the study of endothelial differentiation (21, 22). The tube formation assay was modified from the method described by Gu et al. (23). The wells of four-chamber slides (Nunc, Naperville, IL) were each coated with 50 µL Matrigel containing 6 ng VEGF and 1 ng bFGF. Approximately 30,000 endothelial cells in 0.5 mL medium were seeded into each chamber. After 1 h of incubation, another 0.5 mL medium with control medium, manumycin, paclitaxel, or manumycin plus paclitaxel were added to each well. After incubation for 24 h at 37 C, the slides were fixed in formalin, stained with hematoxylin-eosin, and photographed. The experiments were performed in triplicate.
Statistical analysis
Significance of differences among multiple groups was assessed using one-way ANOVA. The null hypothesis was accepted if P < 0.05.
To analyze the xenograft data, the data for each cell line and each
treatment group were modeled using linear and quadratic growth curves.
Differences between the curves were assessed using mixed model,
repeated measures ANOVA techniques. For each cell line, an overall
analysis was conducted using all treatment groups to provide general
control over type 1 error (experimentwise 
= 0.05). Follow-up
analyses were performed on specific a priori hypotheses. In
each analysis, linear only and linear and quadratic models were fitted
to the data, and the significance of each component was assessed. Where
both linear and higher order models fit the data, the simpler linear
model is reported. Differences in the growth curves were assessed using
Wald 
2 statistics. Because growth curve
analysis requires specification of the underlying repeated measures
covariance structure, both compound symmetry and autoregressive (order
1) covariance structures were tested. The covariance structure with the
largest Akaike information criterion value was used for the final
analysis. Data analyses were performed using BMDP statistical software
(BMDP Statistical Software, Inc., Cork, Ireland).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The in vivo antineoplastic effect of the combination of manumycin and paclitaxel was investigated using the Hth-74 and KAT-18 cell lines xenografted in nude mice. The mice were randomized into four groups (four animals in each group). Each animal in the control group received two ip placebo injections. Each animal in the manumycin group received one ip placebo injection and one ip injection of manumycin (7.5 mg/kg). Each animal in the paclitaxel group received one ip placebo injection and one ip injection of paclitaxel (20 mg/kg). Each animal in the manumycin plus paclitaxel group received ip injections of manumycin (7.5 mg/kg) and paclitaxel (20 mg/kg). The mice received injections twice weekly, and tumor sizes were measured as described above.
Compared with placebo, manumycin and paclitaxel each had a significant
antitumor effect against Hth-74 xenografts (Fig. 1A
; Wald 2
statistics for growth curves, P < 0.05) and the KAT-18
xenografts (Fig. 1B; Wald 2 statistics for
growth curves, P < 0.05). Manumycin plus paclitaxel
showed significantly more inhibitory effect than paclitaxel alone
against the Hth-74 (Fig. 1A; P < 0.05) and the KAT-18
xenografts (Fig. 1B; P < 0.05). The combination also
showed significantly more inhibitory effect than manumycin alone
against the KAT-18 xenografts (Fig. 1B; P < 0.05). The
combination was probably more inhibitory than manumycin alone against
the Hth-74 xenografts (Fig. 1A; P = 0.16), although
significance did not reach the traditional level of P
0.05 (24, 25). When the data from the Hth-74 xenografts
treated with either paclitaxel or manumycin alone were combined in a
group that had been treated with one drug, single agent treatment was
significantly less inhibitory than combination treatment
(P < 0.05). Therefore, the combination of manumycin
and paclitaxel had an improved antineoplastic effect in vivo
than manumycin or paclitaxel alone.
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Manumycin inhibited angiogenesis in vivo
We evaluated the effects of manumycin and paclitaxel on
tumor angiogenesis because manumycin-treated tumors looked paler than
both control and paclitaxel-treated tumors (Fig. 1C
). To determine
whether manumycin, paclitaxel, or a combination of both could block the
angiogenic effects of VEGF and bFGF, mice treated with the drugs were
assessed for formation of new vessels in SQ Matrigel implants
containing VEFG and bFGF and compared with placebo-treated control
mice. The hemoglobin concentrations in Matrigel implants from mice
treated with manumycin or manumycin plus paclitaxel were significantly
lower than those in controls (Fig. 2A;
one-way ANOVA, P < 0.05). There was no significant
difference in the hemoglobin concentration in the Matrigel implants
from the control mice and the mice treated with paclitaxel only.
Sections of Matrigel implants stained with Masson’s Trichrome stain
showed that manumycin and manumycin plus paclitaxel significantly
decreased the vessel area in the Matrigel implants, whereas paclitaxel
alone did not affect vessel area when compared with the control value
(Fig. 2B; one-way ANOVA, P < 0.05). Analysis of
hematoxylin-eosin-stained Matrigel implant sections showed that
treatment with manumycin or manumycin plus paclitaxel significantly
decreased the number of nuclei per microscopic field of the Matrigel,
but not with paclitaxel alone (Fig. 2C; one-way ANOVA,
P < 0.05). Therefore, manumycin inhibited angiogenesis
in vivo, but paclitaxel did not (at the particular dosing
regimen used).
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The effect of manumycin on the production of a major angiogenic
factor VEGF by ATC cells was investigated. Treatment with manumycin and
paclitaxel alone or in combination produced no detectable change in
VEGF in ARO and KAT-4 cells under normoxic culture conditions (data not
shown). Under hypoxic conditions, 16-h treatment of manumycin and
manumycin plus paclitaxel decreased the amount of VEGF in KAT-4 and ARO
cells (Fig. 3). Immunostaining of KAT-4
xenograft samples also revealed that manumycin decreased VEGF (Fig. 4). Therefore, manumycin decreased the
VEGF in ATC cells under hypoxic conditions.
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Next, we focused attention on the vascular components.
Angiogenesis involved proliferation, migration, and differentiation of
the endothelial cells. HUVECp and HMVECad were treated with various
concentrations of manumycin (up to 54 µmol/L) and paclitaxel (up to
22 µmol/L) alone or in combination. Both manumycin alone and
paclitaxel alone inhibited the viability and proliferation of
endothelial cells (Fig. 5). Unlike the
ATC cells (15), the combination of manumycin and
paclitaxel did not result in an enhanced decrease in viability in the
endothelial cells tested.
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The effects of manumycin and paclitaxel on the ability of
endothelial cells to migrate were also studied. Manumycin and
paclitaxel concentrations were chosen such that the viability of the
cells was not compromised. Manumycin (0.5 µmol/L) did not inhibit
migration of HUVECp, but paclitaxel (1.1 µmol/L) inhibited the
migration without killing the cells (Fig. 6A). Similarly, manumycin (1 µmol/L)
did not inhibit the migration of HMVECad, but paclitaxel (1.1 µmol/L)
inhibited the migration without killing the cells (Fig. 6B).
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HMVECad were seeded into four-chamber slides coated with Matrigel.
After incubation for 24 h at 37 C, the endothelial cells organized
into a network and formed tubular structures (Fig. 7, upper left panel). In
the presence of 2 µmol/L manumycin or 1.1 µmol/L paclitaxel alone,
the endothelial cells were not significantly affected (Fig. 7, upper right panel and lower left
panel, respectively). Interestingly, in the presence of both
manumycin and paclitaxel, the linear structures of the network were
disrupted or disorganized (Fig. 7, lower right
panel). As combining manumycin and paclitaxel did not
enhance cytotoxicity in HMVECad (Fig. 5A), this enhanced inhibition of
endothelial cell network formation could not be explained only by
enhanced cytotoxicity.
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Pharmacokinetics can affect both the therapeutic efficacy as well as the toxicity profile of a drug. As angiogenesis and tumor growth are continuous processes, one would predict that sustained delivery of manumycin might result in better antineoplastic efficacy than intermittent administration. Therefore, we investigated the antineoplastic efficacy and toxicity of sustained delivery of manumycin alone and in combination with paclitaxel.
The effect of slow release manumycin pellets was tested in mice bearing
KAT-4 xenografts. In a control experiment, one or two placebo pellets
were implanted into nude mice, and the KAT-4 xenografts grew at the
same alarming rate as with ip placebo treatment (data not shown). This
confirmed that the excipients in the manumycin slow release pellet had
no antineoplastic activity. The growth curve of the KAT-4 xenografts in
mice that received one manumycin slow release tablet (cumulative dose,
0.875 mg/21 days) was significantly different from the tumor growth
curve in nude mice that received a placebo tablet (Fig. 8A; P < 0.05). Thus,
sustained delivery of manumycin produced a significant antineoplastic
effect against the KAT-4 cells.
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). The control group received sc implantation of two placebo pellets
and ip injection of vehicle. The paclitaxel group received sc
implantation of two placebo pellets and ip injection of paclitaxel (20
mg/kg). The manumycin group received sc implantation of two manumycin
pellets (cumulative dose, 1.75 mg/21 days) and ip injection of vehicle.
The manumycin plus paclitaxel group received sc implantation of two
manumycin pellets and ip injection of paclitaxel. The tumor growth
curves demonstrated that both manumycin sc slow release
(P < 0.05) and paclitaxel ip (P <
0.05) had significant antineoplastic activity compared with the control
group. The tumor growth curve of the manumycin plus paclitaxel group
was significantly better than that of the group receiving paclitaxel
alone (P < 0.05). Probably, the tumor growth curve of
the manumycin plus paclitaxel group was also better than manumycin
alone (P = 0.08) although the significance did not
reach the traditional level of P 0.05 (24, 25). Thus, the combination of paclitaxel with sustained delivery
of manumycin provided improved antineoplastic effect compared with
single drug treatment. This finding was similar to the results obtained
with intermittent ip injections of manumycin and paclitaxel.
To test whether sustained delivery of manumycin has better
antineoplastic activity than intermittent ip injection of manumycin,
nude mice xenografted with KAT-4 cells received ip paclitaxel and
either ip manumycin (cumulative dose, 1.08 mg/21 days) or one slow
release manumycin pellet (cumulative dose, 0.875 mg/21 days). The tumor
growth curve of the manumycin pellet plus paclitaxel group was
significantly better than the ip manumycin plus paclitaxel group (Fig. 8C; P < 0.05). Thus, sustained delivery of manumycin
produced more antineoplastic effect against the KAT-4 cells at a lower
cumulative dose than intermittent bolus injection of manumycin.
The body weight of the mice in these groups did not change significantly (data not shown), just as in the experiments in which both drugs were given ip (data not shown). Complete blood counts and liver function tests were also monitored, and no toxicity was observed (data not shown). Thus, sustained delivery of manumycin did not result in increased toxicity in mice.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We evaluated the combination manumycin and paclitaxel for the treatment of ATC, and we found that the combination of manumycin and paclitaxel enhanced the antitumor effects of both drugs against ATC (15). In this paper we present additional in vivo data from nude mice bearing the KAT-18 and Hth-74 xenografts, which demonstrated that the combination of manumycin and paclitaxel had greater antineoplastic effect than did either drug alone. The aggressive growth of the KAT-18 cell line was typical of ATC and was very similar to that of the ARO and KAT-4 cell lines (15). The Hth-74 cell line showed less aggressive growth, and chemotherapy led to tumor regression. The toxicity data indicated that manumycin, paclitaxel, and their combination did not have significant toxic effects in mice. A liver enzyme panel demonstrated a slight rise in lactate dehydrogenase in all groups (including the control group). This slight rise in lactate dehydrogenase was due to slight hemolysis in the blood collection process. Manumycin plus paclitaxel can inhibit ATC without toxicity and, therefore, would support the idea of using manumycin plus paclitaxel in clinical trials after more detailed toxicity studies.
Angiogenesis is an important step in cancer proliferation and metastasis (16). When we dissected ATC xenografts from nude mice, we observed that the manumycin-treated tumors were paler than the tumors not treated with manumycin. A hypothesis that can explain this observation is that manumycin inhibits angiogenesis. This hypothesis was supported by our experiments with Matrigel implants in mice. Treatment of the mice with the same manumycin dose and dosing schedule as in the xenograft experiments decreased the hemoglobin content, the number of endothelial cells, and the vascularity in the Matrigel implants. Given the same proangiogenic stimuli (i.e. the same concentration of bFGF and VEGF in the same volume of Matrigel), manumycin treatment inhibited vascular development. Our results corroborated and extended the findings of Gu et al. (23), who showed that a peptidomimetic FTI, A-170634, decreased vascularization in and around xenografted HCT116 colon cancer cells.
VEGF is an important mitogen for angiogenesis (16). The effects of manumycin and paclitaxel on VEGF in ATC were evaluated in both tumor xenografts and tissue culture. Manumycin decreased the amount of VEGF detected by immunoblotting in two ATC cell lines (ARO and KAT-4) under hypoxic conditions. Although paclitaxel by itself did not affect the level of VEGF, paclitaxel appeared to enhance the inhibitory effect of manumycin. The level of VEGF in KAT-4 xenografts also was lower in the manumycin-treated animals. However, the combination of paclitaxel and manumycin did not enhance inhibition of VEGF expression in the xenografts. In a previous study oncogenic activated tyrosine kinases and Ras proteins induced a 6- to 16-fold increase in VEGF messenger ribonucleic acid, and VEGF messenger ribonucleic acid became more stable (28). The up-regulation of VEGF by activated H-ras in HaCaT cells can be reversed by an FTI (29). Feldkamp et al. (30) found that a peptidomimmetic FTI, L-744832, inhibited the synthesis and secretion of VEGF in the astrocytoma cell line U373 under hypoxic conditions. Gu et al. (23) found that another peptidomimetic FTI, A-170634, decreased VEGF secretion from HCT116 colon cancer cells. Including our results, suppression of VEGF by three FTIs has been demonstrated in four cell lines (ARO, KAT-4, U373, and HCT116), and further investigation may be warranted to determine whether suppression of VEGF by FTIs is a general phenomenon.
Endothelial cells are the source of new blood vessels, and they have a remarkable ability to migrate, proliferate, and differentiate. We focused on the effect of manumycin and paclitaxel on the endothelial cells. Both manumycin and paclitaxel inhibited proliferation and viability of endothelial cells at concentrations lower than those required to inhibit ATC cells. At subcytotoxic doses, migration of HUEVCp and HMVECad was not inhibited by manumycin, but was inhibited by paclitaxel. The conclusion that paclitaxel inhibited migration of endothelial cells is based on the fact that a 24-h incubation with 1.1 µmol/L paclitaxel did not affect the viability of endothelial cells. Belotti et al. (31) reported that paclitaxel demonstrated antiangiogenic activity. Paclitaxel inhibited the proliferation and migration of endothelial cells in our in vitro studies, but paclitaxel did not significantly inhibit angiogenesis in vivo with the dosage and dosing regimen used in our xenograft studies. Gu et al. (23) reported that a peptidomimetic FTI, A-170634, decreased HUVEC capillary structure formation in Matrigel. We found that both manumycin and paclitaxel were capable of inhibiting HMVECad capillary structure formation in Matrigel. Our results also showed that the combination of manumycin and paclitaxel, at doses that produced minimal inhibitory effects when either drug was applied alone, enhanced the inhibition of HMVECad tubular structure formation in Matrigel.
Angiogenesis as well as tumor growth are continuous processes. Given tolerable toxicity, a stable level of the antineoplastic or antiangiogenic drug may be desirable. Continuous infusion of paclitaxel in the treatment of cancer is under investigation (32). Our data demonstrated that sustained delivery of manumycin resulted in improved antineoplastic activity in vivo at a lower cumulative dose than intermittent bolus injections of manumycin. The improvement in antineoplastic effect by the combination of paclitaxel and manumycin was preserved when intermittent injection of manumycin was changed to sustained delivery by implantation of a slow release manumycin pellet. Sustained delivery of manumycin alone or in combination with paclitaxel did not result in increased toxicity in the mice. Therefore, sustained delivery of manumycin is more desirable than intermittent administration. This information may prove to be important in the design of future clinical trials.
In conclusion, our study showed that manumycin inhibits ATC in vivo without toxicity. The combination of manumycin and paclitaxel is more effective than single agent treatment in vivo without increased toxicity. Sustained delivery of manumycin resulted in improved antineoplastic activity than intermittent bolus injections. Manumycin and paclitaxel affected different aspects of tumor angiogenesis, and inhibition of tumor angiogenesis played a significant role in the antineoplastic effect of manumycin plus paclitaxel in vivo in addition to direct cytotoxicity to ATC cells.
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Footnotes |
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2 Present address: Department of Head and Neck Surgery, Cancer
Center, Sun Yat-Sen University of Medical Sciences, Guangzhou,
People’s Republic of China.
Received October 2, 2000.
Revised December 8, 2000.
Accepted December 13, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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V. Poulaki, C. S. Mitsiades, C. McMullan, D. Sykoutri, G. Fanourakis, V. Kotoula, S. Tseleni-Balafouta, D. A. Koutras, and N. Mitsiades Regulation of Vascular Endothelial Growth Factor Expression by Insulin-Like Growth Factor I in Thyroid Carcinomas J. Clin. Endocrinol. Metab., November 1, 2003; 88(11): 5392 - 5398. [Abstract] [Full Text] [PDF]
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M. Braga-Basaria and M. D. Ringel Beyond Radioiodine: A Review of Potential New Therapeutic Approaches for Thyroid Cancer J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 1947 - 1960. [Abstract] [Full Text] [PDF]
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H.-L. Yang, J.-X. Pan, L. Sun, and S.-C. J. Yeung p21 Waf-1 (Cip-1) Enhances Apoptosis Induced by Manumycin and Paclitaxel in Anaplastic Thyroid Cancer Cells J. Clin. Endocrinol. Metab., February 1, 2003; 88(2): 763 - 772. [Abstract] [Full Text] [PDF]
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