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Identification of Biologically Active Factors in Ionizing Radiation Regulated Secretome: Potential Role of Placental Growth Factor Tamara Kazimova1, Martin Pruschy1 1 Department of Radiation Oncology, University Hospital Zurich, 8091 Zurich, Switzerland Ionizing radiation (IR) leads to DNA damage and genome instability. In addition, IR also leads to stress responses in tumor cells by activating signal transduction pathways and inducing secretion of numerous auto- and paracrine factors. As part of an exhaustive IR-dependent secretome analysis which was previously performed in our laboratory, placental growth factor (PlGF) was identified to be secreted in response to IR. It is an N-glycosylated, homodimeric protein and belongs to vascular endothelial growth factor (VEGF)-family. There are four splice isoforms of the protein and they bind to VEGFR1 and NRP1. PlGF expression is low to undetectable in most tissues in healthy subjects, but becomes significantly up regulated in disease. In this project, several tumor cell lines with differential genetic backgrounds were investigated in response to increasing doses of IR. PlGF level was increased in conditioned media (CM) derived from A549 cells in a time and dose-dependent way within the first 24h. PlGF expression was already upregulated after 4 h of irradiation with 5 and 10 Gy in these cells and remained upregulated up to 24 hours. Interestingly, in NCI-H125 lung adenocarcinoma cells increased expression regulation could only be observed after 24 hours. Next, PlGF was genetically targeted using PlGF-directed siRNA, where downregulation was observed at 48 hours and up to 96 hours after siRNA-treatment. CM was collected from unirradiated and irradiated, PlGF depleted cells (siPlGF) and control cells (siLuc). HUVECs were incubated with these differential CM for 20 minutes and cell lysates were analyzed by western blotting. Interestingly, CM derived from irradiated control cells (siLuc) increased the pERK-level in the endothelial cells which was suppressed when incubated with CM derived from PlGF-depleted cells. Control ELISA was performed on these conditioned media and demonstrated lower PlGF level in CM derived from PlGF depleted cells. In conclusion, PlGF which has so far not been investigated in response to IR, might play a relevant role for the radiation response on the level of tumor angiogenesis.
