Comparison of the effects of carbon ion and photon irradiation on the angiogenic response in human lung adenocarcinoma cells.

PURPOSE: Radiotherapy resistance is a commonly encountered problem in cancer treatment. In this regard, stabilization of endothelial cells and release of angiogenic factors by cancer cells contribute to this problem. In this study, we used human lung adenocarcinoma (A549) cells to compare the effects of carbon ion and X-ray irradiation on the cells' angiogenic response. METHODS AND MATERIALS: A549 cells were irradiated with biologically equivalent doses for cell survival of either carbon ions (linear energy transfer, 170 keV/µm; energy of 9.8 MeV/u on target) or X-rays and injected with basement membrane matrix into BALB/c nu/nu mice to generate a plug, allowing quantification of angiogenesis by blood vessel enumeration. The expression of angiogenic factors (VEGF, PlGF, SDF-1, and SCF) was assessed at the mRNA and secreted protein levels by using real-time reverse transcription-PCR and enzyme-linked immunosorbent assay. Signal transduction mediated by stem cell factor (SCF) was assessed by phosphorylation of its receptor c-Kit. For inhibition of SCF/c-Kit signaling, a specific SCF/c-Kit inhibitor (ISCK03) was used. RESULTS: Irradiation of A549 cells with X-rays (6 Gy) but not carbon ions (2 Gy) resulted in a significant increase in blood vessel density (control, 20.71 ± 1.55; X-ray, 36.44 ± 3.44; carbon ion, 16.33 ± 1.03; number per microscopic field). Concordantly, irradiation with X-rays but not with carbon ions increased the expression of SCF and subsequently caused phosphorylation of c-Kit in endothelial cells. ISCK03 treatment of A549 cells irradiated with X-rays (6 Gy) resulted in a significant decrease in blood vessel density (X-ray, 36.44 ± 3.44; X-ray and ISCK03, 4.33 ± 0.71; number of microscopic field). These data indicate that irradiation of A549 cells with X-rays but not with carbon ions promotes angiogenesis. CONCLUSIONS: The present study provides evidence that SCF is an X-ray-induced mediator of angiogenesis in A549 cells, a phenomenon that could not be observed with carbon ion irradiation. Thus, in this model system evaluating angiogenesis, carbon ion irradiation may have a therapeutic advantage. This observation should be confirmed in orthotopic lung tumor models.

EphA2 blockade enhances the anti-endothelial effect of radiation and inhibits irradiated tumor cell-induced migration of endothelial cells.

BACKGROUND: EphA2 tyrosine kinase plays an important role in tumor angiogenesis, but whether targeting this pathway can affect response to ionizing radiation (IR) remains unknown. METHODS: We investigated, using a soluble EphA2-Fc chimera, whether EphA2 inhibition could sensitize A549 and MCF-7 tumor cells, as well as human umbilical vein endothelial cells (HUVEC) and dermal microvascular endothelial cells (HDMEC), to IR. RESULTS: EphA2-Fc resulted in a greater response of endothelial cells (EC) to IR than either treatment alone. EphA2-Fc significantly increased apoptosis and decreased clonogenic survival, tube formation and migration in irradiated EC after stimulation with vascular endothelial growth factor (VEGF), without an affecting their proliferation. No difference in proliferation or survival was found in A549 and MCF-7 tumor cells. In a co-culture model, EphA2-Fc inhibited an irradiated A549 cell-induced increase in EC migration. VEGF supplementation, as well as condiotioned medium from irradiated A549 cells, phosphorylated EphA2 in EC. The latter was abrogated by EphA2-Fc. CONCLUSIONS: EC were most sensitive to a combination of EphA2 inhibition and radiotherapy. The induction of paracrine growth factors and activation of EphA2 in EC suggest a protective mechanism that tumors probably use to attenuate IR-induced antivascular effects. Our data justify further investigation to explore targeting EphA2 in tumor radiosensitivity in vivo.

TIAR and TIA-1 mRNA-binding proteins co-aggregate under conditions of rapid oxygen decline and extreme hypoxia and suppress the HIF-1α pathway.

T-cell intracellular antigen (TIA)-1 and TIA-1-related protein (TIAR) are mRNA-binding proteins that can aggregate within granules under specific stress conditions. In this study, we analyzed TIAR/TIA-1 aggregation under different hypoxic conditions, and studied the effects on the hypoxia-inducible factor (HIF)-1α in different cancer cell lines. Under acute and pronounced hypoxic conditions TIAR/TIA-1 co-aggregated to granules and positive co-staining with eIF3η marker suggested these to represent stress granules. In parallel, HIF-1α expression was blocked in cells displaying TIAR/TIA-1 granules. Silencing of TIAR and TIA-1 caused upregulation of HIF-1α expression, as demonstrated by western blot, immunocytochemistry and HIF-1-dependent reporter gene expression. Additionally, a critical region of the 3' end of the untranslated HIF-1α mRNA with possible adenosine-uridine-rich elements (AREs) was coupled to the luciferase reporter gene, causing downregulation of expression. Employing this reporter construct, inhibition of TIAR by siRNA attenuated the inhibitory cis-effect of this ARE-sequence. Furthermore, immunohistochemical analysis of A549 cell tumor xenografts revealed a nearly complementary expression of HIF-1α and TIAR reflecting the control of HIF-1α expression by TIAR as revealed in the cell culture studies. In sum, rapid and severe hypoxia caused co-aggregation of TIAR/TIA-1 and these proteins suppressed HIF-1α expression.

Irradiation-dependent effects on tumor perfusion and endogenous and exogenous hypoxia markers in an A549 xenograft model.

PURPOSE: Hypoxia is a major determinant of tumor radiosensitivity, and microenvironmental changes in response to ionizing radiation (IR) are often heterogenous. We analyzed IR-dependent changes in hypoxia and perfusion in A549 human lung adenocarcinoma xenografts. MATERIALS AND METHODS: Immunohistological analysis of two exogenously added chemical hypoxic markers, pimonidazole and CCI-103F, and of the endogenous marker Glut-1 was performed time dependently after IR. Tumor vessels and apoptosis were analyzed using CD31 and caspase-3 antibodies. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) and fluorescent beads (Hoechst 33342) were used to monitor vascular perfusion. RESULTS: CCI-103F signals measuring the fraction of hypoxic areas after IR were significantly decreased by approximately 50% when compared with pimonidazole signals, representing the fraction of hypoxic areas from the same tumors before IR. Interestingly, Glut-1 signals were significantly decreased at early time point (6.5 h) after IR returning to the initial levels at 30.5 h. Vascular density showed no difference between irradiated and control groups, whereas apoptosis was significantly induced at 10.5 h post-IR. DCE-MRI indicated increased perfusion 1 h post-IR. CONCLUSIONS: The discrepancy between the hypoxic fractions of CCI-103F and Glut-1 forces us to consider the possibility that both markers reflect different metabolic alterations of tumor microenvironment. The reliability of endogenous markers such as Glut-1 to measure reoxygenation in irradiated tumors needs further consideration. Monitoring tumor microvascular response to IR by DCE-MRI and measuring tumor volume alterations should be encouraged.