sepharose and cobaltous-chloride

The response of cancer cells to hypoxic conditions found within the interior of a tumor mass is mediated through the hypoxia inducible factor (HIF) cascade and is thought to promote metastasis. However, given their distant proximity from blood vessels as compared to normoxic cells at the vascularised tumor periphery, it is uncertain if these cells can migrate through the tumor mass to gain access. Hypoxia was simulated by exposure to cobalt chloride or deferoxamine in normal (MCF10A) and cancerous [estrogen receptor (ER)-ve (pII), and ER +ve (YS1.2/ EII)] cells. In this report, HIF1α expression and localization was measured using western blotting, ELISA, and immunofluorescence, cell proliferation by MTT assay, motility and invasion by wound healing, live cell imaging, matrigel and co-culture in chambered slides. We found that the expression and nuclear translocation of HIF1α was significantly elevated by hypoxia, which inhibited cell proliferation, but significantly increased motility of pII cells and their penetration into and through a dense layer of adjacent EII cells, as well as their selective emergence out of a co-culture. These data suggest that endocrine resistant pII cancer cells, having undergone epithelial to mesenchymal transition are able to penetrate through other cell layers, with possible enhancement in response to hypoxia.

Topics: Basement Membrane; Breast Neoplasms; Cell Hypoxia; Cell Line, Tumor; Cell Movement; Cobalt; Coculture Techniques; Deferoxamine; Epithelial-Mesenchymal Transition; Estrogens; Female; Gene Expression Regulation, Neoplastic; Gene Silencing; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; MCF-7 Cells; Neoplasm Invasiveness; Neoplasm Proteins; Neoplasms, Hormone-Dependent; Receptors, Estrogen; RNA, Small Interfering; Sepharose

To present a novel marker-flange, addressing source-reconstruction uncertainties due to the artifacts of a titanium intracavitary applicator used for magnetic resonance imaging (MRI)-guided high-dose-rate (HDR) brachytherapy (BT); and to evaluate 7 different MRI marker agents used for interstitial prostate BT and intracavitary gynecologic HDR BT when treatment plans are guided by MRI.. Seven MRI marker agents were analyzed: saline solution, Conray-60, copper sulfate (CuSO4) (1.5 g/L), liquid vitamin E, fish oil, 1% agarose gel (1 g agarose powder per 100 mL distilled water), and a cobalt-chloride complex contrast (C4) (CoCl2/glycine = 4:1). A plastic, ring-shaped marker-flange was designed and tested on both titanium and plastic applicators. Three separate phantoms were designed to test the marker-flange, interstitial catheters for prostate BT, and intracavitary catheters for gynecologic HDR BT. T1- and T2-weighted MRI were analyzed for all markers in each phantom and quantified as percentages compared with a 3% agarose gel background. The geometric accuracy of the MR signal for the marker-flange was measured using an MRI-CT fusion.. The CuSO4 and C4 markers on T1-weighted MRI and saline on T2-weighted MRI showed the highest signals. The marker-flange showed hyper-signals of >500% with CuSO4 and C4 on T1-weighted MRI and of >400% with saline on T2-weighted MRI on titanium applicators. On T1-weighted MRI, the MRI signal inaccuracies of marker-flanges were measured <2 mm, regardless of marker agents, and that of CuSO4 was 0.42 ± 0.14 mm.. The use of interstitial/intracavitary markers for MRI-guided prostate/gynecologic BT was observed to be feasible, providing accurate source pathway reconstruction. The novel marker-flange can produce extremely intense, accurate signals, demonstrating its feasibility for gynecologic HDR BT.

Topics: Brachytherapy; Catheters; Cobalt; Copper Sulfate; Feasibility Studies; Female; Fiducial Markers; Fish Oils; Humans; Iothalamate Meglumine; Magnetic Resonance Imaging, Interventional; Male; Phantoms, Imaging; Prostatic Neoplasms; Radiotherapy Dosage; Radiotherapy Planning, Computer-Assisted; Sepharose; Sodium Chloride; Titanium; Tomography, X-Ray Computed; Uncertainty; Uterine Cervical Neoplasms; Vitamin E

We have investigated inhibitory mechanisms of hypoxic activation of HIF-1alpha by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1alpha activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel-Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1alpha, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl(2) or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl(2)-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl(2) was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

Topics: Blotting, Western; Cell Line; Cobalt; DNA Fragmentation; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutaric Acids; Luciferases; Nitric Oxide; Oxygen; Procollagen-Proline Dioxygenase; Protein Binding; Protein Structure, Tertiary; Sepharose; Signal Transduction; Sodium Chloride; Time Factors; Transcription Factors; Transcription, Genetic; Transfection