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Experimental Therapeutics |
Laboratory of Biology and Therapy of Metastasis, Department of Oncology, Mario Negri Institute for Pharmacological Research, 24125 Bergamo, Italy
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Unlike antiangiogenic agents, aimed at preventing vessel formation, the vascular-targeting agents aim to compromise the integrity and functionality of already existing tumor vessels, leading to shutdown of the tumor vascular system and consequent tumor cell death (4)
. Vascular targeting is made possible by the structural, phenotypic, and functional differences between vessels in tumor and normal tissues (5, 6, 7)
. Endothelial cells in tumors are exposed to a peculiar environment (low oxygen tension, low pH, presence of cytokines, and growth factors) and are undergoing angiogenesis. Hence, they differ from quiescent endothelial cells in terms of functional status and gene expression, including the expression of antigenic determinants. It is possible to exploit these changes for therapeutic purposes in two distinct vascular-targeting approaches by: (a) directing therapeutic agents to endothelial cells within tumors or (b) using agents that selectively affect tumor endothelial cells (i.e., tumor necrosis factor 
, flavone acetic acid and its analogue dimethylxanthenone acetic acid, and tubulin-binding agents; Refs. 4
and 8, 9, 10
).
Microtubules are an interesting target for vascular-targeting agents. Compounds that affect microtubule dynamics act as antiangiogenic agents, because they affect endothelial cell functions crucial for this process (motility, invasion, attachment, alignment, and proliferation; Refs. 11, 12, 13 ). Microtubule depolymerizating agents, including colchicine, podophyllotoxin, and Vinca alkaloids, cause damage to the existing vasculature of tumors (4) . However, the narrow therapeutic window of these agents (vascular-targeting activity is achieved only at doses approaching or exceeding their maximum tolerated dose) has prevented their development as vascular-targeting agents. Recently, microtubule-destabilizing agents such as combretastatin A-4 (14) have been described, which have activity against tumor vasculature at doses significantly lower than the maximum tolerated dose, and, therefore, these agents have the potential to exploit this effect therapeutically.
The colchicine derivative ZD6126 is a novel water-soluble phosphate pro-drug. It is converted in vivo into N-acetylcolchinol (ZD6126 phenol), which binds to the colchicine-binding site on tubulin, and causes disruption of microtubules. In animal models, ZD6126 selectively induces tumor vascular damage and massive tumor necrosis at well-tolerated doses (15) . ZD6126 is currently in early phase clinical trial. The aim of this study was to investigate the effects of this vascular-targeting antitumor agent on endothelial cells in vitro and on neo-formed vessels in vivo.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cells.
HUVECs,3
isolated and cultured as described (12)
, were used between the third and fifth passages. HMVEC-Ls from Clonetics (BioWhittaker, Walkersville, MD) were cultured in Endothelial Basal Medium. NIH-3T3 (murine fibroblasts) and MDA-MB-435 (human breast carcinoma cells) were cultured in DMEM 10% fetal bovine serum. SKOV-3 (human ovarian carcinoma cells) and A-10 (rat smooth muscle cells) were cultured in RPMI 1640 10% fetal bovine serum, 2 mM L-glutamine, and 2 mM sodium pyruvate.
Analysis of Cell Morphology.
Nontissue culture 96-well plates (Microtest; Becton Dickinson, Bedford, MA) were coated with 5 µg/ml (625 ng/cm2) fibronectin (Chemicon, Temecula, CA), collagen I (Becton Dickinson), and collagen IV (Sigma, St. Louis, MO) in PBS or with gelatin 1% (Life Technologies, Inc., Paisley, Scotland) for 2 h at 37°C. After washing in PBS, nonspecific sites were blocked with 1% BSA in PBS (30 min at 37°C). Cells (2.5 x 104 cells/well, unless indicated) in DMEM 0.1% BSA were added and let adhere for 4 h at 37°C. Adherent cells were then exposed to ZD6126, N-acetylcolchinol, or vehicle for 40 min (three to six wells for each condition). Wells were washed with DMEM 0.1% BSA to remove detached cells, stained with crystal violet (0.5% in 20% methanol), rinsed with water, and air dried. Cells were analyzed by inverted light microscopy (IX70; Olympus Optical Co., Tokyo, Japan) and computer image analysis (Image Pro-Plus 4.5; Media Cybernetics, LP). The degree of cell spreading was evaluated as the cell area (area of the plate covered by the cells, normalized to the number of adherent cells). To evaluate the number of adhered cells, the stain was eluted with a 1:1 solution of ethanol; 0.1 M sodium citrate and absorbance at 540 nm was read.
Proliferation Assay.
Cells (4 x 103 cells/well) were plated in 96-well plates in complete medium. After 24 h, N-acetylcolchinol was added (0.001–100 µM). After 1 h, cells were washed and incubated for an additional 3 days. Alternatively, the cells were incubated with the compound for 72 h. Cells were stained with crystal violet, and absorbance was measured as for the adhesion assay. Data are the percentage of control proliferation and the IC50, the drug concentration that causes 50% inhibition of cell proliferation.
Immunofluorescence Analysis of the Cytoskeleton.
HUVECs were grown on 1% gelatin-coated glass coverslips for 3–5 days, incubated for 40 min with N-acetylcolchinol or vehicle in DMEM 0.1% BSA, and fixed and permeated with cold absolute ethanol for 10 min at -20°C. Fixed cells were washed and incubated at room temperature for 1 h with antibodies against ß-tubulin (T4026; Sigma), 1:200. After washings, cells were incubated with 1:200 FITC-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) together with the F-actin-binding rhodamine-phalloidin (25 IU/ml; Biomolecular Probes, Leiden, the Netherlands). Coverslips were then washed and mounted with N-propyl gallate in 50% (volume for volume) glycerol and 0.1 M Tris-HCl (pH 8). Samples were analyzed at fluorescence microscopy (IX70; Olympus) with U-HNIBA filter for ß-tubulin and U-HWIG2 for F-actin.
Cord Disruption Assay.
HUVECs (2 x 104 cells/well) were plated in 96-well plates on a thick layer of Matrigel (Becton Dickinson; 10 mg/ml, 60 µl/well) and let align for 24 h. N-acetylcolchinol or vehicle was added to the formed cords and left for 24 h. Pictures were taken at the indicated times (0.5–24 h) after compound addition.
In Vivo Angiogenesis Assay.
The Matrigel plug assay was used (12)
. Briefly, basic FGF (500 ng/pellet) was embedded in Matrigel (12.5 mg/ml, 0.5 ml) and injected s.c. in C57BL/6N mice (Charles River, Calco, Italy). ZD6126 (200 mg/kg, i.p., single injection) or vehicle (PBS + 5% NaCO3) was given to mice 1, 5, or 22 h before necropsy (day 7 after Matrigel injection, n = 6). One h before necropsy, mice were injected i.v. with 100 µl of FITC-conjugated Griffonia Simplicifolia Isolectin-B4 (0.25 mg/ml; Vector, Burlingame, CA). The Matrigel plug was removed and frozen. Perfused, FITC-positive vessels were analyzed with a laser confocal microscope (InSight Plus; Meridian Instruments, Inc., Okemos, MI) with Argon 488 nm emission source.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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10 times more potent than the pro-drug ZD6126 (data not shown), suggesting a limited conversion of the pro-drug in our experimental conditions in the absence of serum. The subsequent in vitro experiments were performed only with N-acetylcolchinol.
HUVECs were left to adhere to different extracellular matrix components for 4 h and then exposed for 40 min to N-acetylcolchinol. Treated cells retracted and assumed a rounded morphology, indicated by the reduced cell area (Fig. 1, A and B)
. Similar effects were observed when cells were plated on fibronectin, gelatin, type I collagen, and type IV collagen (IC50 ranged from 0.51 to 0.83 µM; Fig. 1
). The change in cell shape was only partially associated with detachment of the cells from the substratum, because N-acetylcolchinol caused only 
20% cell detachment (data not shown). When added to endothelial cells while they were adhering to the substrata, the compound only marginally impaired attachment (data not shown), excluding an effect on cell–substrate recognition/binding.
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. N-acetylcolchinol (1 µM) caused complete depolymerization of microtubules in 40 min and induced the formation of actin stress fibers across the cell body (Fig. 1)
. Many cells retracted and exhibited blebbing of the plasma membrane (Fig. 1)
. The effect of N-acetylcolchinol of endothelial cell morphology was more prominent on scattered, nonconfluent cells (in three experiments, mean IC50 was 0.07 ± 0.01 µM), whereas confluent, quiescent cells were less sensitive (IC50 = 0.62 ± 0.2 µM).
Alteration of cell morphology occurred at concentrations of N-acetylcolchinol that did not impair endothelial cell proliferation. When HUVECs were exposed for 1 h to N-acetylcolchinol, inhibition of cell proliferation was observed only at concentrations much higher (100–1000-fold) than those required to alter cell shape (Table 1)
. Only at longer exposure times (72 h) was an antiproliferative effect observed (data not shown).
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. Nonendothelial cells were generally less sensitive to the compound in terms of cell shape changes compared with endothelial cells (Table 1)
. N-acetylcolchinol caused marginal changes in the morphology of smooth muscle cells A-10, which were also less sensitive to the antiproliferative activity of the compound (Table 1)
. NIH-3T3 fibroblasts showed an intermediate behavior between HUVEC and A10 (Table 1)
. The morphology of the two tumor cell lines tested (SKOV-3 and MDA-MB-435) was only partially affected by N-acetylcolchinol (41 and 37% reduction in cell area, respectively), although the compound had an antiproliferative activity comparable with endothelial cells (Table 1)
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The modifications induced by N-acetylcolchinol on endothelial cell morphology (both HUVECs and HMVEC-Ls) were reversible, and cells reverted to the original shape by 3 h after removal of the compound. Recovery of cell shape was faster (1 h) when cells were treated with low concentrations of the compound (0.1–1 µM), but even after treatment with higher concentrations, no significant permanent modification of the cell shape was apparent (Fig. 2)
. Colchicine, a related tubulin-targeting agent, induced similar changes in endothelial cell morphology, although in this case, changes were not reversible (data not shown).
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1 µM N-acetylcolchinol to formed cords rapidly disrupted the integrity of the network (Fig. 3, A and B)
. The effect was reversible because, 24 h later, cells appeared to have re-established the original structures (Fig. 3, C and D)
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presented a strong angiogenic response, with numerous functional, perfused neo-vessels, particularly abundant and presenting a larger caliber at the periphery of the pellet. Treatment of mice with ZD6126 (200 mg/kg i.p.) caused an almost complete shutdown of the vessels, which, 1 h after treatment, were no longer perfused by FITC-isolectin B4 (Fig. 3F)
. H&E staining and immunostaining for the endothelial cell-specific antigen CD31 showed that vascular structures were still present in parallel pellets (data not shown). Similar to our observations on in vitro cords, also in this in vivo model, the effect was reversible, and 22 h after treatment, vessels in Matrigel were again perfused (Fig. 3H)
. The phenomenon of reperfusion appeared primarily at the periphery of the pellet. |
DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In vitro, N-acetylcolchinol causes retraction of endothelial cells, disrupting the tubulin cytoskeleton and inducing actin stress fibers and membrane blebbing. Cooperation between microtubules and the actin cytoskeleton is crucial in the control of cell shape, adhesion, contraction, and motility (16) . Microtubules directly control actin filament organization by locally modulating the activity of the small GTPases of the Rho family. In particular, microtubule-disrupting agents induce guanine nucleotide exchange factor GEF-H1-mediated activation of Rho, which triggers stress fiber formation (17) . Combretastatin A4 causes Rho-mediated endothelial cell retraction, membrane blebbing, and assembly of stress fibers (18) . Therefore, it is possible that morphological changes and cytoskeletal modifications caused by N-acetylcolchinol might involve GEF-H1/Rho activation.
The ability of tubulin-binding agents to cause rapid endothelial cell retraction is thought to underlie their vascular-targeting activity. In vivo, in a tumor setting, endothelial cell retraction may lead to the increased permeability, exposure of the basement membrane, platelet activation, and coagulation. Accordingly, in a panel of tumor models in vivo, ZD6126 induced rapid (30–60 min) changes in central tumor capillaries with focal loss of endothelial cells, exposure of the basal lamina, accumulation of platelets and fibrin, thrombus formation, and congestion of tumor vessels (15) . By 24 h, extensive central necrosis of the tumor, with only a thin viable rim of tumor cells at the periphery, is observed (15 , 19) .
After injection of a single, well-tolerated dose (61 mg/kg) to mice, ZD6126 is rapidly converted to N-acetylcolchinol (peak concentrations at 10 min). Thereafter, the plasma concentration of N-acetylcolchinol declines (half-life of 
1 h), and 1 h after administration, plasma concentrations are 2 µg/ml (5.6 µM; Ref. 15
). The plasma profile of N-acetylcolchinol in mice (consistent with the conditions active in vitro on endothelial cell morphology), together with the rapid elimination of the compound from the plasma, may further rule out a significant effect on cell proliferation, which requires higher concentrations and/or longer times of exposure.
N-acetylcolchinol alters more effectively the shape of nonconfluent endothelial cells than confluent, quiescent endothelial cells. This might depend on differences in the stability of the actin cytoskeleton, in the strength of cell–cell interactions or in the expression of tubulin isotypes, tubulin post-translational modifications, or microtubule-associated proteins between confluent and nonconfluent cells. This difference in sensitivity suggests that the compound is more likely to affect immature endothelial cells (such as those in tumor vessels) rather than mature, quiescent cells of vessels in normal tissues. In agreement, preclinical studies showed activity of ZD6126 on tumor vessels without similar effects on vessels in normal tissues (15) .
The effect of N-acetylcolchinol on endothelial cell morphology is rapid and reversible, because within 1–3 h after removal of the compound, the cells reverted to their original shape. Although several mechanisms might contribute to the reversibility of the compound activity, this behavior is in agreement with the tubulin-binding kinetics of this class of compounds: (a) a rapid binding to tubulin and (b) a very short dissociation half-life (20) . This property represents an improvement compared with other microtubule-destabilizing agents, such as colchicine, whose narrow therapeutic window has been ascribed to the pseudo-irreversible binding to tubulin (4 , 14) .
The reversibility of the effect of ZD6126/N-acetylcolchinol on endothelial cell morphology, in vitro cord disruption, and in vivo vessels occlusion in the Matrigel model suggests a potential therapeutic margin in the use of this compound. However, these findings also suggest that the damage to tumor vascular induced by this compound may not be permanent and that, especially at the periphery of a tumor, vessels may rapidly recover their functionality. This observation parallels the in vivo evidence that single treatment with ZD6126, although causing central tumor necrosis, may induce only a modest tumor growth delay in animal models (15) . Repeated single agent administration or combination with conventional therapies offer promising therapeutic potential for ZD6126. Indeed, recent preclinical studies have confirmed the increased antitumor activity of multiple doses of ZD6126 (15) and of combinations of this compound with chemotherapy and radiotherapy regimens (15 , 19) .
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported partially by AstraZeneca (Alderly Park, Macclesfield, Cheshire, United Kingdom) and grants from the Italian Foundation for Cancer Research and the Italian Association for Cancer Research. 
2 To whom requests for reprints should be addressed, at Mario Negri Institute for Pharmacological Research, Via Gavazzeni 11, 24125 Bergamo, Italy. Phone: (39) 035 319888; Fax: (39) 035 319331; E-mail: giavazzi{at}marionegri.it
3 The abbreviations used are: HUVEC, human umbilical vein endothelial cell; FGF, fibroblast growth factor; HMVEC-L, human lung microvascular endothelial cell.
Received 9/30/02. Accepted 1/31/03.
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