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The Antiangiogenic Property of Docetaxel Is Synergistic with a Recombinant Humanized Monoclonal Antibody against Vascular Endothelial Growth Factor or 2-Methoxyestradiol but Antagonized by Endothelial Growth Factors¹

Indiana University, Departments of Medicine [C. J. S., K. D. M., G. W. S.] and Biostatistics [ D. K. H., J. S.], and Walther Oncology Center [S. E. S., S. N.], Indianapolis, Indiana 46202

	ABSTRACT

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Numerous chemotherapeutic agents have been shown to have an inhibitory effect on endothelial cell proliferation and migration, and tubule formation. In this study, we examined the antiangiogenic activity of docetaxel. Docetaxel inhibited endothelial cell proliferation and tubule formation in vitro in a dose-dependent fashion. Docetaxel treatment also inhibited angiogenesis in an in vivo Matrigel plug assay. The endothelial stimulating factors, vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor are able to protect endothelial cells from the antiangiogenic properties of docetaxel. This protective effect can be overcome by a recombinant humanized monoclonal antibody directed against VEGF in both in vitro and in vivo models. Similarly, combination of docetaxel with the antiangiogenic agent 2-methoxyestradiol also overcomes the protective effect of VEGF in both in vitro and in vivo models. These data suggest that microenvironmental factors (e.g., local release of VEGF and basic fibroblast growth factor) could play a role in decreasing the antiangiogenic effects of docetaxel, whereas agents such as 2- methoxyestradiol and recombinant humanized monoclonal antibody directed against VEGF may reverse this protective effect.

	INTRODUCTION

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Angiogenesis (new blood vessel development) is vital to the growth and metastasis of human cancers, and as such it represents a potential therapeutic target. Numerous agents, both novel agents and traditional cytotoxics, have been shown to inhibit angiogenesis (1) . Paclitaxel is a semisynthetic taxane that is cytotoxic by binding to tubulin and preventing microtubule disassembly. This results in the disruption of the dynamic equilibrium of mitosis and cell division and induces apoptosis (2) . More recently, paclitaxel has been found to inhibit angiogenesis in both in vitro and in vivo models (3, 4, 5) . Docetaxel is a related taxane, derived from Taxus baccata, with a similar mode of action (6) . To date, there is little information regarding the antiangiogenic effects of docetaxel. This report examines the antiangiogenic effects of docetaxel in vitro.

If the antiangiogenic activity of the taxanes represents part of their antitumor activity, it is reasonable to ask why this activity has not led to the eradication of human solid tumors. This report examines the role of the proangiogenic factors VEGF³ and bFGF in inducing resistance to the antiangiogenic effects of docetaxel. It also examines means by which this novel form of drug resistance might be overcome.

	MATERIALS AND METHODS

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Materials.
HUVECs (Clonetics, San Diego, CA) were cultured in EGM-2 medium (Clonetics) and harvested after having undergone no more than five passages. Docetaxel powder (provided by Aventis Pharmaceuticals, Parsippany, NJ) in 100% alcohol without polysorbate-80, rhuMAb-VEGF in PBS (provided by Genentech, South San Francisco, CA), and 2ME₂ in DMSO (provided by EntreMed, Rockville, MD) were added at varying clinically relevant concentrations to a proliferation and a capillary formation assay. VEGF (Chemicon International, Temecula, CA), and bFGF (R&D Systems Minneapolis, MN) were added to the assays in doses that have been shown in vitro to induce the maximal amount of HUVEC endothelial cell proliferation (7 , 8) . Thrombin, bovine fibrinogen, and aprotinin (all from Sigma Chemical Co, St. Louis MO) were used for the formation of a fibrin clot. Microcarrier beads consisting of a thin layer of denatured collagen chemically coupled to a matrix of cross-linked dextran (175 µm, Cytodex 3; Amersham Pharmacia, Biotech AB, Uppsala, Sweden) were used as the base for the capillary formation. This assay has been shown to induce capillaries with identifiable lumens (9) . The MTS/PMS system (Promega, Madison WI) was used to assess cell viability for the proliferation assay.

Proliferation.
HUVECs were plated in a 96-well U-bottomed plate (Becton Dickinson Labware, Franklin Lakes, NJ) at a concentration of 10,000 cells/50 µL of medium and incubated in 5% CO₂ at 37°C for 48 h. Varying drug concentrations in 50 µl of medium were added to the medium, and this mixture was added to each well within 1 h of the HUVECs being seeded. The proliferation experiments were performed with and without stimulation by the addition of VEGF (60 ng/ml) and bFGF (20 ng/ml). These factors in high doses were chosen to partially simulate the tumor microenvironment. Colorimetric readings were obtained using the MTS/PMS system and an ELISA plate reader. The readings obtained for each concentration tested were from an average of eight wells. Each experiment was expressed as a percentage of the solvent control and completed at least three times with consistent results. The results presented are an average of at least three experiments.

Endothelial Cell Capillary Formation.
Microcarrier beads (200 mg) suspended in PBS were autoclaved and then added to HUVECs at a concentration of 30 HUVECs per microcarrier bead. Microcarrier beads and cells were added to a siliconized Petri dish and rocked at 37°C in 5% CO₂ for 48 h. The HUVEC-coated microcarrier beads were transferred to a fibrin clot solution. Fibrinogen was dissolved at 2.5 mg/ml in PBS with 0.15 units/ml of aprotinin. Approximately 20 HUVEC-coated microcarrier beads were added to each well of a 12-well plate, and then thrombin (0.625 units/ml) was added to form a gelatinous clot. Medium (1.5 ml) with 1% human serum and aprotinin (0.15 units/ml) was added to the top of each clot. The addition of VEGF (60 ng/ml) and bFGF (20 ng/ml) was required to ensure robust capillary formation. There was minimal capillary formation without stimulation; therefore, all results reported are with stimulation. The drugs to be tested were also added to the top layer. Capillary formation was then quantified after 4 days: every capillary greater than the radius of the bead was scored, and the average number of tubules for each bead per well was determined. The results were expressed as a fraction of the positive control. The experiments were repeated at least three times, and the results presented are the average of at least three experiments.

Assessment of Multiple Drug Effects.
The Chou and Talalay (10) method for assessing the combined effect of multiple drugs based on the median effect principle was used in conjunction with the "Calcusyn" program (Biosoft, Cambridge, United Kingdom). Dose-response curves were generated for each drug alone to determine the median effect plots. Variable ratios of drug concentrations and mutually exclusive equations were used to determine the CIs. This analysis allows drugs with different mechanisms of action to be assessed in combination and for different interactions at low and high doses to be observed if present. A CI <1 denotes synergy (more than additive), a CI of 1 demonstrates summation (additive), and a CI >1 indicates antagonism (less than additive).

In Vivo Analysis.
Low growth factor Matrigel (Becton Dickinson, Bedford, MA) was supplemented with 10⁴ cancer cells/ml. The breast cancer cell line MCF-7 and a genetically modified MCF-7 variant with transfection of the VEGF gene (both a kind gift from Dr. Francis Kern, Molecular Oncology, Southern Research Institute, Birmingham, AL) were used. The MCF-7 contains neither VEGF receptor, and any effects noted are effects of the cytokine on the surrounding vasculature. Estrogen pellets were inserted 72 h prior to injection of 0.3 ml Matrigel into both flanks of nude mice. The right flank had the parental MCF-7 cell line, and the left had the VEGF transfected cell line. Treatment was commenced at day 7 after injection of the Matrigel. Treatment groups consisted of 10 mice that were treated in the following manner: (a) solvent control, 3 mg/kg of docetaxel given i.p. on days 7 and 10; (b) 0.25 mg/kg rhuMAb-VEGF given i.p. on days 7–14; (c) 20 mg/kg 2ME₂ by oral gavage on days 7–14; (d) 3 mg/kg docetaxel plus 0.25 mg/kg rhuMAb-VEGF on days 7 and 10; (e) 3 mg/kg of docetaxel on days 7 and 10 plus 20 mg/kg 2ME₂ on days 7–14. On the 14th day, each plug was resected, weighed, and immersed in 10 ml of distilled water per g of Matrigel plug. The mixture was placed in a shaking water bath for 24 h. After 24 h, 100 µl were mixed with 100 µl of Drabkin’s reagent (Sigma) for 15 min at room temperature and then placed in a 96-well plate. The hemoglobin concentration was determined by absorbance readings on a plate reader. Two absorbance readings were recorded per sample with the average reported for each. The result for each treatment group was reported as the average from the 10 mice in each cohort. The presence of statistical differences was evaluated by the two-sample two-sided t test without log transformation.

	RESULTS

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In Vitro Analysis
Docetaxel as an Antiangiogenic
Docetaxel inhibited HUVEC proliferation in a dose-dependent manner (Fig. 1) . HUVECs incubated in the presence of stimulation required a higher concentration of docetaxel (38.0 ng/ml) to inhibit proliferation by 50% (IC₅₀) compared with the unstimulated cells (7.7 ng/ml). It was also noted that stimulation antagonized the effect of docetaxel at all doses >0.1 ng/ml because the CIs were >10 (Fig. 1) . Much lower concentrations of docetaxel were required to inhibit capillary formation. When docetaxel was dosed as a single agent, it inhibited capillary formation in a dose-dependent fashion starting at 0.01 ng/ml with an IC₅₀ of 0.27 ng/ml (Fig. 2) .

View larger version (28K): [in this window] [in a new window]   Fig. 1. Dose-dependent inhibition of HUVEC proliferation by docetaxel: protective effect of stimulation is overcome by rhuMAb-VEGF. Doc, docetaxel; St, stimulation; antiV, rhuMAb-VEGF.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Dose-dependent inhibition of capillary formation by docetaxel is synergistic with rhuMAb-VEGF.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Dose-dependent inhibition of HUVEC proliferation by 2ME2 stimulation does not induce resistance and is synergistic with docetaxel.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Dose-dependent inhibition of capillary formation by 2ME2 is synergistic with docetaxel.

Combination Proapoptotic Agents (Docetaxel Plus 2ME₂).
We checked for the presence of synergy with docetaxel and 2ME₂ in the presence of stimulation. Synergy was noted for the combination of 1 ng/ml docetaxel plus 0.1, 0.5, and 1 µM 2ME₂ (Fig. 3) . However, slight antagonism was observed when 2ME₂ was dosed at 5 and 10 µM with 1 ng/ml docetaxel. In the capillary formation assay, the combination of low dose (0.1 ng/ml) docetaxel was synergistic with 0.1, 0.5, 1, 2.5, and 5 µM 2ME₂ (Fig. 4) .

In Vivo Correlation
The in vitro findings were confirmed in the in vivo experiments. Angiogenesis was assessed by measuring the amount of hemoglobin in the Matrigel plugs and recorded as an absorbance reading. The control cohort (parental MCF-7) gave an absorbance reading of 4.43 (Table 1) . The amount of angiogenesis in the VEGF-transfected cohort was 1.7-fold greater (P = 0.001) with an absorbance reading of 7.56. The amount of angiogenesis in the docetaxel treated cohort was 54% of control (P = 0.0048). The amount of angiogenesis in the mice with the VEGF-transfected MCF-7 cells was decreased to 57% of that in the untreated mice with the transfected cells (P = 0.0004). It is of note that the amount of angiogenesis in the mice with the VEGF-transfected cell line treated with docetaxel (absorbance reading of 4.36) did not differ statistically (P = 0.921) from the observations in the untreated mice with the parental MCF-7 cell line (absorbance reading of 4.42). This confirms the in vitro findings that showed that excess VEGF protects the endothelial compartment from docetaxel.

Combination therapy was also evaluated. The rhuMAb-VEGF plus docetaxel combination was able to impact on the amount of angiogenesis in the mice with VEGF-transfected cells as effectively if not slightly better than docetaxel alone given to mice with parental MCF-7 (P = 0.07). In other words, rhuMAb-VEGF was able to overcome the resistance to docetaxel induced by VEGF. Docetaxel plus 2ME₂ was very effective therapy, with the amount of angiogenesis being 17% of control in the mice with parental MCF-7 (P = 0.0009) and 27% of control when the same therapy was given to mice with VEGF-transfected MCF-7 cells (P = 0.0006).

	DISCUSSION

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Docetaxel has in vitro antiangiogenic properties that are dose dependent and achievable at plasma concentrations routinely achieved in humans. This statement is based on the fact that docetaxel dosed at 100 mg/m² every 3 weeks in humans results in a serum concentration >50 ng/ml for 10 h (11) . It is also of note that in vitro cytotoxic doses of docetaxel against cancer cells are in the same range as the doses required to inhibit endothelial cell proliferation. Specifically, the IC₅₀ of docetaxel for a variety of cancer cell lines has been reported to be <10 ng/ml (12) . Docetaxel has also been shown to inhibit endothelial cell (HUVEC) migration in a modified Boyden chamber assay with an IC₅₀ of 0.01 nM (13) . Therefore, docetaxel has in vitro antiangiogenic properties against three endothelial cell functions (proliferation, migration, and capillary formation). Moreover, the antiangiogenic IC₅₀s are below the serum levels achieved in humans and are similar to the in vitro cytotoxic IC₅₀s against cancer cells.

There is also clinical evidence supporting the notion that taxanes are antiangiogenic. Namely, low weekly doses of taxanes have been shown to induce tumor regression in some patients with tumors refractory to paclitaxel administered every 3 weeks (14) . These responses to low, frequent taxane exposure may occur as result of inhibition of tumor angiogenesis. However, the fact that cancers are not eradicated by this schedule of taxanes implies that the vascular compartment may be resistant to the antiangiogenic properties of taxanes.

Although low doses of docetaxel inhibit HUVEC proliferation and capillary formation in vitro, cancers are able to grow and divide in patients with ample vasculature. The disconnect between the in vitro and the clinical data suggests that the vasculature in the tumor microenvironment may be "protected" or that regrowth is not dependent on the development of new vasculature. The data presented here infer that VEGF and bFGF, two substances secreted by tumor cells and present in the tumor microenvironment, are able to alter the phenotype of HUVECs and protect them from docetaxel. Taken together, these data support the findings that intracellular signals are "switched" on by growth factors and that these signals can be blocked by rhuMAb-VEGF. Specifically, VEGF has been shown to prevent apoptosis by activating p44 mitogen-activated protein kinase, c-jun-NH2-kinase, phosphoinositide 3-OH kinase, and Akt pathways as well as by inducing Bcl-2 and inhibitors of apoptosis (7 , 15 , 16) . Fibroblast growth factor has also been shown to induce Bcl-2 and survivin in endothelial cells (7 , 17) . It has also been observed that endothelial cells derived from the tumor microenvironment express P-glycoprotein, whereas HUVECs do not and that the latter were markedly more sensitive to the cytotoxic effects of paclitaxel (18) .

The data presented here also suggest that the survival properties induced by growth factors can be overcome with either an agent that directly blocks these survival factors (rhuMAb-VEGF) or with low doses of an antiangiogenic agent with proapoptotic capabilities (2ME₂; Ref. 19 ). 2ME₂ is an estrogen metabolite with very little estrogenic activity that has been observed in only a few systems. It is also antiproliferative in tumor and endothelial cell lines in both in vitro and in vivo assays (20 , 21) . The observation that stimulation affected the antiproliferative effects of docetaxel but not those of 2ME₂ suggests that these agents induce apoptosis by different mechanisms. The fact that synergy was observed at low doses of both docetaxel and 2ME₂ suggests that apoptosis is induced most efficiently at low doses. In contrast, the antagonism noted at higher doses of 2ME₂ with 1 ng/ml docetaxel suggests that these drugs may interfere with the apoptotic capabilities of one another. This may be relevant when determining the doses of drugs to use when designing clinical trials.

The in vivo data presented correlate with the in vitro findings. It must be pointed out that the model used does not differentiate well between an antitumor and a direct antiangiogenic effect. However, it does evaluate the effect of treatment on the tumor microenvironment. The model also evaluates the effect of the paracrine release of VEGF on the efficacy of treatment in inhibiting angiogenesis. Moreover, because MCF-7 cells have neither VEGF receptor 1 nor 2, the changes induced by this cytokine are most likely the result of its effect on the vasculature. With this understanding, we concluded that docetaxel can inhibit angiogenesis in vivo and that excess VEGF can impair this capability. This "resistance" phenomenon was less apparent with the other proapoptotic agent, 2ME₂. It was also noted that rhuMAb-VEGF or 2ME₂ was able to enhance the ability of docetaxel to decrease the amount of angiogenesis in the tumor microenvironment. The augmentation of the effect was seen in both the unstimulated and stimulated environments.

Three recent reports have shown in vivo that a combination of low, frequent dose chemotherapy plus an agent that specifically targets the endothelial cell compartment is able to eradicate or control tumor growth much more effectively than the cytotoxic alone. The agents used were low-dose cyclophosphamide plus TNP-470 (22) , vinblastine plus an antibody against VEGF receptor-2 (23) , and carboplatin plus rhuMAb-VEGF (24) . The data presented here provide rationale for combining low doses of docetaxel with rhuMAb-VEGF or 2ME₂.

In conclusion, there is an increasing amount of data suggesting that a combination of a low, frequent dose of a cytotoxic agent that targets the tumor cell and the vascular compartment in combination with an agent that inhibits angiogenesis may be a fruitful area for future clinical research.

	ACKNOWLEDGMENTS

 
We thank Joanne Dunn for technical assistance.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by Breast Cancer Research Foundation, The Catherine Peachey Fund, and Aventis Pharmaceuticals.

² To whom requests for reprints should be addressed, at Route 473, Indiana Cancer Pavilion, 535 Barnhill Drive, Indianapolis, IN 46202.

³ The abbreviations used are: VEGF, vascular endothelial growth factor; bFGF, basic fibroblast growth factor; HUVEC, human umbilical venous endothelial cell; rhuMAb-VEGF, recombinant humanized monoclonal antibody directed against VEGF; 2ME₂, 2 methoxyestradiol; CI, combination index.

Received 7/10/00. Accepted 2/ 8/01.

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				B. P. Schneider and K. D. Miller Angiogenesis of Breast Cancer J. Clin. Oncol., March 10, 2005; 23(8): 1782 - 1790. [Full Text] [PDF]

				G. Gasparini, R. Longo, M. Fanelli, and B. A. Teicher Combination of Antiangiogenic Therapy With Other Anticancer Therapies: Results, Challenges, and Open Questions J. Clin. Oncol., February 20, 2005; 23(6): 1295 - 1311. [Abstract] [Full Text] [PDF]

				K. Podar and K. C. Anderson The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications Blood, February 15, 2005; 105(4): 1383 - 1395. [Abstract] [Full Text] [PDF]

				H.-P. Gerber and N. Ferrara Pharmacology and Pharmacodynamics of Bevacizumab as Monotherapy or in Combination with Cytotoxic Therapy in Preclinical Studies Cancer Res., February 1, 2005; 65(3): 671 - 680. [Abstract] [Full Text] [PDF]

				G.-Z. Han, Z.-J. Liu, K. Shimoi, and B. T. Zhu Synergism between the Anticancer Actions of 2-Methoxyestradiol and Microtubule-Disrupting Agents in Human Breast Cancer Cancer Res., January 15, 2005; 65(2): 387 - 393. [Abstract] [Full Text] [PDF]

				J. L. Ricker, Z. Chen, X. P. Yang, V. S. Pribluda, G. M. Swartz, and C. Van Waes 2-Methoxyestradiol Inhibits Hypoxia-Inducible Factor 1{alpha}, Tumor Growth, and Angiogenesis and Augments Paclitaxel Efficacy in Head and Neck Squamous Cell Carcinoma Clin. Cancer Res., December 15, 2004; 10(24): 8665 - 8673. [Abstract] [Full Text] [PDF]

				M. Cristofanilli and G. N. Hortobagyi Breast Cancer Highlights: Key Findings from the San Antonio Breast Cancer Symposium: A U.S. Perspective Oncologist, July 1, 2004; 9(4): 471 - 478. [Abstract] [Full Text] [PDF]

				A. B. Sandler, D. H. Johnson, and R. S. Herbst Anti-Vascular Endothelial Growth Factor Monoclonals in Non-Small Cell Lung Cancer Clin. Cancer Res., June 15, 2004; 10(12): 4258S - 4262S. [Abstract] [Full Text] [PDF]

				R. S. Herbst and A. B. Sandler Non-Small Cell Lung Cancer and Antiangiogenic Therapy: What Can Be Expected of Bevacizumab? Oncologist, June 1, 2004; 9(suppl_1): 19 - 26. [Abstract] [Full Text] [PDF]

				H. S. Rugo Bevacizumab in the Treatment of Breast Cancer: Rationale and Current Data Oncologist, June 1, 2004; 9(suppl_1): 43 - 49. [Abstract] [Full Text] [PDF]

				L. G. Estevez and W. J. Gradishar Evidence-Based Use of Neoadjuvant Taxane in Operable and Inoperable Breast Cancer Clin. Cancer Res., May 15, 2004; 10(10): 3249 - 3261. [Abstract] [Full Text] [PDF]

				S. D. Baker, M. Zhao, C. K. K. Lee, J. Verweij, Y. Zabelina, J. R. Brahmer, A. C. Wolff, A. Sparreboom, and M. A. Carducci Comparative Pharmacokinetics of Weekly and Every-Three-Weeks Docetaxel Clin. Cancer Res., March 15, 2004; 10(6): 1976 - 1983. [Abstract] [Full Text] [PDF]

				S. T. Nawrocki, B. Sweeney-Gotsch, R. Takamori, and D. J. McConkey The proteasome inhibitor bortezomib enhances the activity of docetaxel in orthotopic human pancreatic tumor xenografts Mol. Cancer Ther., January 1, 2004; 3(1): 59 - 70. [Abstract] [Full Text]

				E. Esteban, L. Gonzalez de Sande, Y. Fernandez, N. Corral, J Fra, I. Muniz, J. M. Vieitez, I. Palacio, J. L. Fernandez, E. Estrada, et al. Prospective randomised phase II study of docetaxel versus paclitaxel administered weekly in patients with non-small-cell lung cancer previously treated with platinum-based chemotherapy Ann. Onc., November 1, 2003; 14(11): 1640 - 1647. [Abstract] [Full Text] [PDF]

				S. M. Plum, A. D. Hanson, K. M. Volker, H. A. Vu, B. K. L. Sim, W. E. Fogler, and A. H. Fortier Synergistic Activity of Recombinant Human Endostatin in Combination with Adriamycin: Analysis of in Vitro Activity on Endothelial Cells and in Vivo Tumor Progression in an Orthotopic Murine Mammary Carcinoma Model Clin. Cancer Res., October 1, 2003; 9(12): 4619 - 4626. [Abstract] [Full Text] [PDF]

K. D. Miller, C. J. Sweeney, and G. W. Sledge The Snark is a Boojum: the continuing problem of drug resistance in the antiangiogenic era Ann. Onc., January 1, 2003; 14(1): 20 - 28. [Abstract] [Full Text] [PDF]