Abstract
This study tests whether the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (L-NNA), combines favorably with ionizing radiation (IR) in controlling squamous carcinoma tumor growth. Animals bearing FaDu and A431 xenografts were treated with L-NNA in the drinking water. IR exposure was 10 Gy for tumor growth and survival studies and 4 Gy for ex vivo clonogenic assays. Cryosections were examined immunohistochemically for markers of apoptosis and hypoxia. Blood flow was assayed by fluorescent microscopy of tissue cryosections after i.v. injection of fluorospheres. Orally administered L-NNA for 24 hrs reduces tumor blood flow by 80% (p<0.01). Within 24 hrs L-NNA treatment stopped tumor growth for at least 10 days before tumor growth again ensued. The growth arrest was in part due to increased cell killing since a combination of L-NNA and a single 4 Gy IR caused 82% tumor cell killing measured by an ex vivo clonogenic assay compared to 49% by L-NNA or 29% by IR alone. A Kaplan-Meyer analysis of animal survival revealed a distinct survival advantage for the combined treatment. Combining L-NNA and IR was also found to be at least as effective as a single i.p. dose of cisplatin plus IR. In contrast to the in vivo studies, exposure of cells to L-NNA in vitro was without effect on clonogenicity with or without IR. Western and immunochemical analysis of expression of a number of proteins involved in NO signaling indicated that L-NNA treatment enhanced arginase-2 expression and that this may represent vasculature remodeling and escape from NOS inhibition. For tumors such as head and neck squamous carcinomas that show only modest responses to inhibitors of specific angiogenic pathways, targeting NO-dependent pro-survival and angiogenic mechanisms in both tumor and supporting stromal cells may present a potential new strategy for tumor control.
Highlights
Nitric oxide synthase (NOS) activity is a key component in a number of survival mechanisms integrated into the autocrine and paracrine nature of tumor cells and supporting stromal cells
L-NNA delays A431 and FADU xenograft growth To determine the effect of L-NNA on the growth of tumors, xenografts were grown unhindered until approximately 150 mm3 in volume, at which point they were divided into four treatment groups: control; 0.5 g/L L-NNA in drinking water from day 0; 10 Gy ionizing radiation (IR) on day 1; or L-NNA from day 0 and 10 Gy IR on day 1
A similar set of experiments was performed with FaDu xenografts to evaluate if the inhibitory effect of L-NNA on tumor growth could be generalized to another tumor
Summary
Nitric oxide synthase (NOS) activity is a key component in a number of survival mechanisms integrated into the autocrine and paracrine nature of tumor cells and supporting stromal cells. In vitro experiments with different cell types have demonstrated that 30–50% of the basal NF-kB activity is sensitive to either NOS inhibitors or dominant negative NOS mutants [6]. These NOS-dependent survival mechanisms are activated by ionizing radiation (IR). (IR) stimulates the activity of eNOS (NOS-3) in tumor endothelial cells resulting in enhanced tumor angiogenesis through RTK-dependent and –independent mechanisms [7,8,9]. In mutant Ras transformed cells, Akt phosphorylation and activation of eNOS results in the Snitrosylation (or oxidation to sulfenic acid) of Ras Cys118, enhancing GTP binding, and thereby stimulating cytoprotective signaling pathways [10]. In vitro experiments have shown that activation of these NF-kB and RTK ‘‘pro-survival’’ mechanisms by IR can be inhibited by the NOS inhibitor NG-nitro-L-arginine (L-NNA) [6,7]
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