Biology contribution
Augmentation of radiation response with the vascular targeting agent ZD6126

https://doi.org/10.1016/j.ijrobp.2005.11.017Get rights and content

Purpose: To examine the antivascular and antitumor activity of the vascular targeting agent ZD6126 in combination with radiation in lung and head-and-neck (H&N) cancer models. The overall hypothesis was that simultaneous targeting of tumor cells (radiation) and tumor vasculature (ZD6126) might enhance tumor cell killing.

Methods and Materials: A series of in vitro studies using human umbilical vein endothelial cells (HUVEC) and in vivo studies in athymic mice bearing human lung (H226) and H&N (squamous cell carcinoma [SCC]1, SCC6) tumor xenografts treated with ZD6126 and/or radiation were performed.

Results: ZD6126 inhibited the capillary-like network formation in HUVEC. Treatment of HUVEC with ZD6126 resulted in cell cycle arrest in G2/M, with decrease of cells in S phase and proliferation inhibition in a dose-dependent manner. ZD6126 augmented the cell-killing effect of radiation and radiation-induced apoptosis in HUVEC. The combination of ZD6126 and radiation further decreased tumor vascularization in an in vivo Matrigel angiogenesis assay. In tumor xenografts, ZD6126 enhanced the antitumor activity of radiation, resulting in tumor growth delay.

Conclusions: These preclinical studies suggest that ZD6126 can augment the radiation response of proliferating endothelial H&N and lung cancer cells. These results complement recent reports suggesting the potential value of combining radiation with vascular targeting/antiangiogenic agents.

Introduction

Cancers of the upper aerodigestive tract represent a major public health problem in the United States and worldwide. Despite progress in modern therapy, only 33% and 11% of patients with advanced lung cancer (the leading cause of cancer death in the United States) survive beyond 1 and 2 years, respectively (1). Novel therapeutic approaches are clearly needed. An emerging strategy with high promise involves antivascular treatment to target either existing tumor vasculature (vascular targeting/disrupting) or the formation of new blood vessels that feed tumors (antiangiogenic therapy) (2). The rationale for these strategies is based on the concept that tumors require blood vessels to grow and metastasize (3). With the knowledge that a substantial proportion of lung and head-and-neck (H&N) cancer patients receive radiation as an integral component of their treatment, it is valuable to examine the capacity of vascular targeting or antiangiogenic agents to modulate radiation response and tumor control. It is anticipated that simultaneous targeting of tumor cells (radiation) and tumor vasculature (vascular targeting/antiangiogenic agents) might enhance tumor cell killing in lung and H&N cancers.

In this study, we examined the antivascular and antitumor activity of ZD6126, a tubulin-binding agent, in combination with radiation in lung and H&N cancer model systems. The vascular targeting agent ZD6126 (AstraZeneca Pharmaceuticals, Macclesfield, UK) is a phosphate prodrug of N-acetylcolchinol (NAC or ZD6126 phenol), which binds to cellular tubulin, leading to destabilization of tubulin cytoskeleton (4, 5). The development of ZD6126 and other tubulin-binding drugs is based on the concept that tumor blood vessels are distinct from vessels found in normal tissues (6). In fact, new tumor vasculature is a somewhat chaotic network of tortuous, immature vessels lacking the support of pericytes seen in normal blood vessels. Furthermore, tumor vessels consist of proliferating, immature endothelial cells that rely on tubulin cytoskeleton to maintain their shape. Therefore, ZD6126 is thought to selectively target tumor endothelial cells and tumor vasculature. Electron microscopic examination of capillaries in ZD6126-treated xenograft tumors revealed loss of endothelial cell cytoplasm, multifocal exposure of basal membranes associated with platelet accumulation, and fibrin deposits (5). ZD6126 treatment leads to extensive necrosis in the central regions, but a rim of viable tumor remains at the periphery, owing to nutritional support from adjacent normal tissue blood vessels that are typically nonresponsive to ZD6126 (2, 5). Because this residual tumor at the periphery is likely to be better oxygenated, it has been hypothesized that ZD6126 and radiation might act in a complementary fashion (i.e., ZD6126 attacks the poorly oxygenated, radioresistant central tumor cell subpopulation, and radiation preferentially kills well-oxygenated proliferating cells at the periphery of the tumor) (7, 8, 9). Recently, combining ZD6126 with radiation was reported to result in greater tumor cell killing when compared with single-agent treatment in several human xenograft models, such as A549 lung carcinoma (10), KHT sarcoma (7, 9), and CH3 mouse mammary carcinoma (11), although not in U87 human glioblastoma xenografts (12). Preclinical studies have also suggested potential benefits of combining radiation with other vascular-disrupting tubulin-binding agents, such as combretastatin (13, 14, 15). To extend our knowledge in this area, we have investigated the antitumor effect of ZD6126 in combination with radiation in lung and H&N tumors, where radiation serves as an integral component of treatment. Specifically, we examined the antitumor activity of ZD6126 in combination with radiation in solid tumor xenograft models and in proliferating endothelial cells to further explore the underlying mechanisms of interaction for ZD6126.

Section snippets

Chemicals

Endothelial growth culture medium (EGM-2) was obtained from Cambrex (Clonetics, East Rutherford, NJ). Cancer cell culture media (Dulbecco modified Eagle medium [DMEM] and Roswell Park Memorial Institute medium [RPMI]) were obtained from Mediatech (Herndon, VA). Matrigel was obtained from Becton Dickinson (Bedford, MA). All other chemicals were purchased from Sigma (St. Louis, MO). ZD6126 was generously provided by AstraZeneca Pharmaceuticals.

Cell lines and cell culture

Human umbilical vein endothelial cells (HUVEC)

Capillary-like network formation

To investigate the effect of ZD6126 on angiogenesis, we performed an in vitro tube formation assay, in which the impact of ZD6126 on HUVEC capacity to form capillary-like networks was examined. In the control wells, HUVEC quickly attached to the reconstituted extracellular matrix (Matrigel). As early as 3 h after seeding, HUVEC mobilized on the gel, spread out, and generated lateral processes to form intercellular tubular connections, creating a capillary-like network (Fig. 1). The endotubes

Discussion

In the present studies, we demonstrate that combination treatment with radiation and ZD6126 is more potent than either agent alone in lung and H&N tumor xenograft model systems. The in vitro data suggest that ZD6126 might influence tumor blood supply by inhibiting the formation of new blood vessels (antiangiogenic mechanism). As seen in the tube formation assay, ZD6126 prevented cell-to-cell interaction and the formation of capillary-like networks at drug concentrations as low as 0.01 μmol/L

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    Supported in part by a preclinical research award from AstraZeneca (P.M.H.), Training Grant CA 87718 from the National Cancer Institute, and an Eastern Cooperative Oncology Group Young Investigator Award (T.H.).

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