At the same time, we compared these biological behaviors with traditional endothelial cell, human umbilical vein endothelial Selleck P505-15 cell (HUVEC) and the original cancer cells. Further, we tried to explore the underlying mechanisms
by detection the expression of some relative genes. Methods Cell culture Human epithelial ovarian carcinoma cell lines SKOV-3 and ES-2 were purchased from American Type Culture Collection (ATCC, Manassas, VA), and were maintained in McCoy’s 5a. Primary human umbilical vein endothelial cells (HUVEC) were isolated from umbilical vein and cultured as described previously  Three-dimensional cultures and hypoxic treatment Thirty microliters of Matrigel (B&D, Bedford, MA) were dropped onto each glass coverslip in a 12-well culture plate and polymerized for 1 h at room
temperature, followed by 30 min’s incubation at 37°C in a humidified 5% CO2 incubator, as described previously . Tumor cells (1 × 104) were seeded onto the three-dimensional gel. The medium supplied with 15% FBS was changed every 36 h. Hypoxic condition was created by flushing 5% CO2 and 95% N2 through a modified chamber (Mitsubishi, Japan), until O2 concentration was reduced to 1%, measured with a Mini oxygen meter. The culture system was sealed and incubated at 37°C . The cells were treated with 50 nM Sirolimus (Sigma, St. Louis, MO) in DMSO to inhibit the role of HIF-1α www.selleckchem.com/products/nvp-bsk805.html under hypoxia when necessary. Proliferation assay For the proliferation assay, 1 × 104 SKOV-3, ES-2 and HUVEC cells, were seeded into a flat bottom 96-well
plate and incubated at 37°C for 3 and 7 d under normoxia or hypoxia (1% O2) respectively, prior to the addition of 20 μL of MTT solution (5 mg/ml in PBS). After incubated for additional 4 h at 37°C, absorbance at 490 nm was measured with a multi-function reader (Tecan GENios, Zurich, Switzerland) to determine cell viability. Cell cycle and apoptosis assay Cell cycle and apoptosis assay were performed on cells with or without hypoxia treatment (for 3 or 7 d) to determine whether hypoxia regulates the growth phase and apoptosis of epithelial ovarian cells. Cells were MYO10 trypsinized and centrifuged at 300 × g (1000 rpm) for 5 min, then resuspended (1 × 106 cells/ml) and fixed with 70% ice-cold ethanol for 30 min, followed by centrifuged, washed and resuspended in 500 μl PBS contained 10 μl of DNase free RNase (final concentration is 1‰). After 30 min incubation, pyridine iodide (PI, 0.05 mg/ml) was added to the solution to incubate for an additional 15 min in the dark and filtered by a nylon mesh to remove cell clusters. The fluorescence of PI was measured using FACS Calibur Flow Cytometer (Becton-Dickinson, San Jose, CA). Cell subpopulations in G0/G1, S and G2/M phases and apoptosis were calculated by gating analysis based on differences in DNA content.