Gene expression of angiogenic factors in A549 cells after carbon-ion and photon irradiation

Radiation therapy is an integral part of the multi disciplinary therapy of lung cancer. Genetic changes and the specific tumor microenvironment favour therapy resistance of tumor cells. Clinical studies suggest that particle irradiation with carbon ions may improve the therapy response of solid tumors compared to photon radiation [1]. Occurrence of therapy resistance is related to secretion of growth and angiogenic factors that stabilize and promote the tumor vasculature [2]. Thus, we analyzed the effects of either carbon ion or photon irradiation on the gene expression of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). VEGF is a well established factor that promotes angiogenesis (extension of existing vessels) and PlGF is a factor that initiates de novo angiogenesis by recruitment of stem cells (vasculogenesis) [3]. Values for relative biological effectiveness after exposure to photon (6 MV-X) or carbon ion (11.4 MeV/u) irradiation were calculated from clonogenic survival experiments according to Puck and Marcus [4]. From these preliminary results we selected 6 Gy for photonand 2 Gy for carbon ion irradiation for the further analyses of the expression of angiogenic factors. The effect of irradiation was confirmed by the typical induction of CDKN1A mRNA in A549 cells (4 h and 24 h) that is observed by both photonand carbon ion irradiation. Quantification of mRNAs for CDKN1A, VEGF and PlGF was performed by realtime RT-PCR (Figure 1). Interestingly, carbon ion irradiation (4 h and 24 h) caused a significant induction of VEGF-mRNA and PlGFmRNA. After photon irradiation a significant increase of VEGF-mRNA and PlGF-mRNA was observed after 4 h whereas no more changes could be demonstrated after 24 h. CDKN1A-mRNAs were significantly increased after photonand carbon ion irraditation at both time points. The expression levels of mRNAs are presented by the ∆Ct values ∆Ct = Ct (gene of interest) – Ct (house keeping gene). As a house keeping gene porphobilinogen deaminase mRNA was used, a gene that is constitutively expressed and not changed after irradiation. A lower ∆Ct indicates a higher mRNA level (a difference of -1 corresponds approximately to a twofold increase between treated and control group). Figure 1. Realtime RT-PCR of VEGF-, PlGFand CDKN1A-mRNAs after carbon ion and photon irradiation (4 h and 24 h). In further experiments, we plan 1) to characterize the effects of irradiation on the protein levels of VEGF and PlGF, 2) to investigate whether the induction of VEGF and PlGF by irradiation is relevant in functional angiogenesis (matrigel plug assay).