Stabilization of hypoxia inducible factor (HIF) alpha
A hypoxia-inducible factor, stable technique, applied in the field of compounds that can be used in these methods
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Embodiment 1
[0252] Example 1 Stabilization of HIFα in vitro cells
[0253] Adenovirus-denatured fetal kidney epithelial cells (293A), cervical epithelial adenocarcinoma cells (HeLa), liver cancer cells (Hep3B), foreskin fibroblasts (HFF), breast epithelial carcinoma cells (MCF7), vein-bearing endothelial cells ( HUVEC), microvascular endothelial cells (HMEC-1), umbilical squamous cell carcinoma cells (SCC-25), lung fibroblasts (HLF), vein endothelial cells (AG10774B) tissues (see, e.g., American Type Culture Collection, ManassaVA ; Qbiogene, the human cells of Carlsbad CA) were planted in 35mm culture dish respectively, at 37 ℃, 20% O 2 , 5%CO 2 Cultured in the environment, the medium is as follows: HeLa cells in Dulbecco's modified Eagle medium (DMEM), 2% fetal bovine serum (FBS); HFF and HLF cells in DMEM, 10% FBS; 293A cells in DMEM, 5% FBS; HUVEC and AG10774B cells in endothelial growth medium (EGM-2; BioWhittaker, Inc., Walkersville MD); HMEC-1 in RPMI 1640, 10% FBS; Hep3B cells in...
Embodiment 2
[0257] Embodiment 2: oxygen consumption effect
[0258] Oxygen-sensing cell culture dishes (BD Bioscience, Bedford MA) contained a spike complex that was more fluorescent under low oxygen conditions. Thus, if oxygen-consuming cells are present in the dish, the fluorescent readout signal is enhanced, which skews the equilibrium toward a state of low oxygen saturation and high fluorescence. Compounds that stabilize HIF by inhibiting hydroxylation are expected to reduce oxygen consumption, ie, oxygen consumption by hydroxylation itself, and / or shift cellular metabolism from aerobic to anaerobic energy.
[0259] Culture adenovirus-denatured fetal kidney epithelial cells (293A) or cervical epithelial adenocarcinoma cells (HeLa) (American type strain collection), and keep them at 37°C and 10% CO 2 Conditions were confluent in medium (high glucose DMEM (Mediatech, Inc., Herndon VA), 1% penicillin / streptomycin mixture (Mediatech), 1% baby calf serum). Cells were collected and resusp...
Embodiment 3
[0262] Example 3: Expression of HIF-regulated genes in vitro
[0263] Vascular endothelial growth factor (VEGF) was analyzed on the opsonizing medium collected from the cell culture fluid prepared as in Example 1 using a QUANTIKINE immunoassay device (R&D) according to the manufacturer's instructions. Such as Figure 4 As shown in A, foreskin fibroblasts (HFF), adenovirus The expression of VEGF in denatured fetal kidney epithelial cells (293A) and liver cancer cells (Hep3B) decreased ( Figure 4 A). Values on the Y axis represent the log of the fold induction relative to the reference 2 value, such that a value of 1 represents a 2-fold induction.
[0264] Alternatively, at 37C and 10% CO 2 Human cells derived from adenovirus-denatured fetal kidney epithelial cells (293A) were cultured in DMEM, 5% FBS, 1% penicillin-streptomycin. After 48 hours, the cells were collected and pooled in a 35mm culture dish. The medium used was conventional medium, and the medium was change...
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