Low LET Radiation-Induced Abscopal Effect and Tumor Recurrence: Nuclear Factor Kappa B (NF-κB) and Tumor Necrosis Factor alpha (TNF-α) Mediated Positive Feedback Mechanism.

The overall objective is to determine whether the surviving cancer cell after radiotherapy can initiate bystander response that result in clonal selection and tumor recurrence at the treatment site. We have two kinds of cell lines: human breast adenocarcinoma cells (MCF-7) and human aortic endothelism cells. First, in the MCF-7 cells we examined whether a bystander effect is initiated by TNF-a through activation of NF-kB which in-turn is triggered by therapeutic doses of radiation exposure, and also whether TNF-a and NF-kB can induce one another through positive feed back mechanisms and maintain a “memory” of the initial irradiation insult for an extended period. Second, between the MCF-7 and HAEC, we demonstrated whether radiation can induce the VEGF secreted by MCF-7, and concordant expression of the VEGFR in the bystander HAEC. MCF-7 cells were exposed to 137 Cs γ ray to a total dose of 2 Gy at a dose rate of 1.06 Gy/min. Both NF-κB DNA-binding activity and the transcriptional activation measured by mobility shift assay and luciferase reporter assay, respectively showed radiation could induce nuclear translocation and transactivate κB-dependent gene expression. The activation was found to be bi-phasic, reached its maximum at 15 min (4.7-folds), decreased to basal level at 16 h, and reached a second phase maximum at 24 h (2.6-folds). Similarly, the levels of TNF-α determined by bioactivity assay revealed a 40% increase in soluble TNF-α in the culture supernatant upon 2 Gy exposure. Incubation of MCF-7 cells with either NF-κB inhibitor isohelenin or TNF-α neutralizing antibody blocked the presence of soluble TNF-α in the culture supernatant and second phase NF-κB activation, respectively. These results clearly indicated the occurrence of positive feed-back cycle between NF-κB and TNF-α through autocrine mechanism. In order to determine the occurrence of a similar mechanism in the non-irradiated bystander MCF-7 cells, supernatant from cells exposed to 2 Gy was added into the non-irradiated cells, and the NF-κB activity of non-irradiated MCF-7 cells increased by 7.2-folds. Next, to examine whether TNF-α is the paracrine signaling mediator in this bystander effect, un-irradiated MCF-7 cells, transiently transfected with NF-κB-luciferase reporter construct, was incubated with the conditioned medium from 2 Gy exposed cells in the presence or absence of TNF-α antibody. The luciferase activity showed a 60% increase in the un-irradiated MCF-7 cells when incubated with medium from irradiated cells in the absence of TNF-α antibody. Molecular analysis of specific angiogensis markers using hAngio-1 probe as a positive control was performed using ribonuclease protection assay. Of the angiogenic RNA molecules identified, the MCF-7 cells expressed VEGF and FGF-2, and the westernblot also showed the VEGF ligand and FGF-2 secreted by MCF-7 incresed by 3.4-folds and 2.6-folds separately after 2Gy exposure. We also co-cultured the irradiated MCF-7 cells with the non-irradiated HAEC, and found VEGFR2 and FGFR1 had corcondant expression increse in HAEC. This mechanism may be responsible for the stimulation of proliferative response, tumor-cell re-growth and subsequent tumor relapse at the treatment site. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2149.