Elsevier

Biochemical Pharmacology

Volume 75, Issue 3, 1 February 2008, Pages 627-638
Biochemical Pharmacology

Antitumor effect of the angiogenesis inhibitor bevacizumab is dependent on susceptibility of tumors to hypoxia-induced apoptosis

https://doi.org/10.1016/j.bcp.2007.09.029Get rights and content

Abstract

Angiogenesis inhibition has been shown to enhance the therapeutic efficacy of cytotoxic chemotherapy in colorectal cancer. The basis of the contribution of this modality has not been defined fully. To determine the potential role of hypoxia-induced apoptosis, we studied a series of colon cancer cell lines with varying susceptibility to hypoxia. We exposed HT29 and HCT116 colon adenocarcinoma cell lines to sublethal periods of hypoxia three times weekly for 40 exposures, and derived cell lines both more resistant (from HT29) and more sensitive (from HCT116) to hypoxia-induced apoptosis. Both hypoxia-derived cell lines demonstrated more rapid growth than the parental lines when implanted subcutaneously in immunodeficient mice. Treatment of tumor-bearing mice with bevacizumab resulted in depletion of tumor microvasculature, upregulation of Hypoxia-inducible factor-1 alpha (HIF-1α), and increased pimonidazole staining, consistent with an anti-angiogenic effect and induction of hypoxia in tumors derived from all cell lines. The proportion of apoptotic cells was increased in all the treated tumors, and was most pronounced in the bevacizumab-treated HCT116-derived cells. The bevacizumab-treated tumors showed growth delay in HT29 and its derivative, and the parental HCT116. In the hypoxia-sensitive HCT116-derived tumors, marked tumor shrinkage and prolonged growth control occurred. Therefore, bevacizumab treatment is an effective inducer of a hypoxic environment, but the resulting cell kill and tumor shrinkage is determined by the susceptibility of the tumor to apoptosis. The induction of apoptosis by hypoxia may contribute to the benefits of such treatment in the clinical setting.

Introduction

The vasculature of solid tumors is often disordered as a consequence of overproduction of factors promoting vessel formation [1]. The degree of angiogenic activity in colorectal cancers is variable, and in many studies is a determinant of tumor aggressiveness and of patient survival [2], [3]. Two studies in colorectal cancer have provided definitive demonstration that targeting angiogenesis may confer therapeutic benefit in cancer [4], [5]. The addition of the anti-vascular endothelial growth factor (anti-VEGF) monoclonal antibody bevacizumab to chemotherapy resulted in improved response rates [4] and survival [4], [5] in patients with advanced disease. The demonstration of the efficacy of combining chemotherapy with bevacizumab prompts interest in the mechanism through which angiogenesis inhibitors exert their effects.

Early studies showed the ability of angiogenesis targeted drugs to cause tumor shrinkage with apoptosis in animal models [6], [7], [8]. Klement et al. demonstrated an effect of continuous low-dose (metronomic) chemotherapy on endothelial cell survival, and showed a sensitizing effect of an anti-VEGF antibody on this cell type [9]. A focus on the disordered vasculature and recognition of increased intratumoral hydrostatic pressure by Jain and co-workers [10], [11], [12] suggested that the effect of bevacizumab with chemotherapy may be to restore order to the vasculature, normalize hydrostatic pressure, and permit enhanced drug delivery to the tumor cells. Consistent with this, Willett and colleagues recently demonstrated that intratumoral hydrostatic pressure of patients with rectal cancer is reduced following treatment with bevacizumab [13]. Jain has emphasized that continued antiangiogenic effects should curtail tumor blood flow and by implication starve the cells of oxygen and nutrients [12]. However the extent of such tumor starvation and the consequences for tumor cell kill have not previously been addressed. The clinical success of antiangiogenic therapy may indeed be viewed as paradoxical, since for many years the focus of therapeutics, particularly in the radiobiological literature has been on eliminating tumor hypoxia, which was well established as a barrier to successful treatment with radiation and alkylating agents. To explore the relationship between inhibition of angiogenesis and the induction of tumor cell kill, we derived a series of colon cancer cell lines with varying susceptibility to hypoxia-induced apoptosis. We showed in vivo that bevacizumab treatment resulted in an increase in hypoxia within the tumor, but that the consequent induction of apoptosis depended on susceptibility to hypoxia-induced apoptosis, and that the apoptotic effect was associated with tumor growth control.

Section snippets

Establishment of hypoxia-conditioned cell lines

The establishment and characterization of hypoxia-conditioned cell lines from HT29 and HCT116 have been described previously [14]. Briefly, the two colon cancer cell lines were exposed to repeated 4h periods of sublethal hypoxia (0.1% O2 for 4 h) followed by return to a standard culture environment (5% CO2, 95% air) to mimic the variable and fluctuating oxygen levels to which tumor cells may be exposed [15]. The treatment was applied thrice weekly, resulting in the generation of a series of cell

Characteristics of the hypoxia-conditioned cell lines

Cell lines were derived by repeated exposure of the colon cancer cell line HT29 (a p53 mutated line, with intact mismatch repair, and a chromosomal instability [CIN+] phenotype) and HCT116 (a p53 wild-type line, with a defect in DNA mismatch repair but no chromosomal instability [CIN]) to 4 h periods of hypoxia thrice weekly, resulting in the generation of cell lines HP40 and HCP40, respectively, each reflecting 40 exposures to hypoxia. The conditioned cell lines manifested stable changes in

Discussion

The introduction of anti-angiogenic therapy for colorectal cancer represents an advance in therapeutics that is expected to extend to other cancers also [26], [27], [28], [29]. Response rates following chemotherapy were increased from 35 to 45% with the addition of bevacizumab in patients with advanced colon cancer, and median survival increased from 15.6 to 20.3 months [4]. These results show that there was incremental benefit, but that the majority of patients still did not respond. In an era

Acknowledgement

This work is supported in part by CA49820 from NCI/NIH/DHHS.

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