Possible roles of nitric oxide in the adhesion of glioma cells to extracellular matrix components after ionizing irradiation

Abstract

Nitric oxide (NO) is a stable free radical in tumors and normal tissue. The generation of free radicals by irradiation may play a role in the reoxygenation of tumor and in the emergence of late toxicity of normal tissue by the interaction with nitric oxide as well as in the tumor progression and metastasis. In this study, we investigated the effect of NO on the adhesion capacity of glioma cells to extracellular matrix components after exposed to γ-irradiation. Three isoforms of NO synthases (NOS) were determined in cells using immunohistochemical staining. The NOS enzymatic activity was measured by determination of NO products using either C14-citrulline measurement or Griess reaction assay. The C6 glioma cells were pretreated with either SNAP, which is an NO donor, or L-NNA, which is an NO synthase inhibitor, followed by irradiation with a total dose of 200, 400 and 800 cGy at a dose rate of 90 cGy/min using Ce-137 irradiator. The adhesion assay was performed in vitro with extracellular matric components including fibronectin, laminin I and collagen IV respectively. Our results showed that 1) The g-irradiation can enhance the endogenous NO production in glioma cells. The enhancement of NO productivity was further achieved to about 3 folds in LPS and IFN-g treated cells indicating the promotion of NO production by ionizing irradiation in glioma cells. 2) At the irradiation dose of 400 and 800 cGy, the inhibition of glioma cell adhesion to ECM by exogenous NO was reduced. Meanwhile, the enhancement of adhesion of glioma cells exposed to irradiation and treated with L-NNA was also abolished in compared to the cells without irradiation. 3) Surviving fraction study for the NO-treated cells under irradiation indicated that 400 cGy is critical for NO-related adhesion since the exogenous NO could decrease surviving fraction to about 50% while inhibition of NO synthase enzyme activity by L-NNA could increase of surviving fraction to about 23%. Our results indicated that ionizing irradiation has an impact on the adhesion of glioma cells and the free radical nitric oxide may have a role as one of the mediators during the tumor metastasis