Kongres w Linz 2-4 wrzesień 2010
14.10.2010
Linz 2010 16th Congress on Alternatives to Animal Testing
Trójmiejska Akademicka Zwierzętarnia Doświadczalna – Centrum Badawczo-Usługowe zaprezentowała poster
Identification of antiangiogenic capability of antitumor agents based on screening for HIF-1 pathway inhibitors: characterization in comparison with in vivo tumor angiogenesis models on the examples from studies on imidazoacridinones
Jolanta Paradziej-Łukowicz,1,2, Anna Skwarska,2, Grażyna Peszyńska-Sularz,1, Jerzy Konopa 2,
1 Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology, Narutowicza St. 11/12, 80-233 Gdansk, Poland
2 Tri-City Central Animal Laboratory – Research and Service Centre of the Medical University of Gdansk, Debinki St. 1, 80-211 Gdansk, Poland
Key words: angiogenesis, imidazoacridinone C-1311, hypoxia, HIF-1, VEGF
Background: Hypoxia-inducible factor-1α (HIF-1α) is a pro-survival transcription factor that particularly promotes tumor angiogenesis by increasing the production of angiogenic factors, such as vascular endothelial growth factor (VEGF). Here we present how simple cell-based approach for the identification of HIF-1 pathway inhibitors was useful in prediction of antiangiogenic capability of imidazoacridinones, a group of DNA-damaging antitumor alkylators. The most promising imidazoacridinone, C-1311, is undergoing phase II clinical trails for breast cancers [2,4].
Materials and methods: Hif-1α DNA-binding activity was determined using ELISA with double-stranded DNA probe encompassing canonical HIF-1α binding site, hypoxia-responsive element (HRE sequence). In vitro studies were conducted on human colon adenocarcinoma HCT116 and mouse melanoma B16/F10 cells. Real-time PCR analysis was applied to determine HIF-1α and VEGF mRNA expression. Secrection of VEGF into conditioned media was examined using ELISA. The mouse Matrigel assay and the alginate beads model with angiogenesis stimulated by encapsulated B16/F10 melanoma cells were used to test the effect imidazoacridinones on angiogenesis in vivo [1,3].
Results: We found that in a cell-free system, C-1311 interfered with HIF-1α binding to an oligonucleotide containing HRE sequence present in VEGF promoter. In hypoxic human colon adenocarcinoma HCT116 and mouse melanoma B16/F10 cells, C-1311 significantly decreased HIF-1α mRNA and protein expression. Consistent with HIF-1α inhibition/depletion, C-1311 (1-10 μM) markedly reduced the level of hypoxia-induced VEGF mRNA expression and protein secretion to extracellular medium. Based on these promising in vitro results, C-1311 was subjected for further analysis in animals. At a single dose (40 mg/kg), C-1311 inhibited angiogenesis in vivo by 70%, monitored as bFGF-stimulated vascularization of Matrigel plugs injected into mice. Of note, the observed decrease in hemoglobin content within Matrigel plugs was similar to that found for 170 mg/kg cyclophosphamide used as a positive control for antiangiogenic effects. Antiangiogenic activity of C-1311 was further confirmed in experiments, where angiogenesis was stimulated by proangiogenic B16/F10 melanoma cells encapsulated in alginate beads and implanted into mice. Importantly, imidazoacridinone C-1310, the second top putative HIF-1α DNA binding inhibitor, as revealed in experiments with HRE probe, also reduced neovascularization of alginate implants in mice, however this effect was less profound than that of the parental C-1311.
Discussion: To conclude, using both cell-free and cell-based approaches, we identified C-1311 as a novel compound, that targets the hypoxia pathway by inhibiting HIF-1α.. Importantly C-1311-mediated inhibition of HIF-1α and VEGF in vitro correlated well with the suppression of angiogenesis in vivo, which emphasizes the fact that simple in vitro screening for HIF-1-targeting drugs with concurrent evaluation of their inhibitory potential may be useful in prediction of antiangiogenic properties of antitumor compounds.
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