Zhu et al. Journal of Experimental & Clinical Cancer Research 2010, 29:124 http://www.jeccr.com/content/29/1/124 Page 2 of 8 mimicry (VM), which indicates the process by which aggressive tumor cells are able to generate not-endothe- lial cell-lined channels delimited by extracellular matrix in vitro [7-9]. That’s the reason why it is difficult to control ovarian cancer with angiogenesis-targeted ther- apy strategies [9] which have no positive effect on such vasculogenesis. Hypoxia is one of the major important factors in angiogenesis descried by Folkman for it is associated with resistance to chemo- and radio-therapies. The development of tissue hypoxia is characteristically observed as malignant tumor rapidly increase in size. Such hypoxic conditions exert selective pressure on can- cer cells, and the ability of tumor cells to survive in a hypoxic microenvironment has been associated with a poor prognosis and resistance to therapy [10]. One of the most critical and best characterized responses to hypoxia is the induction of vascular endothelial growth factor (VEGF), and hypoxia-inducible factor-1 (HIF-1) is a well-established mediator in this process. Our previous studies have demonstrated that the ovarian cancer cells could be induced into endothelial-like cells which have the specific characteristics of endothelial cells at the condition of hypoxia in vivo and in vitro [11-13], in which HIF-1a played a vital role. As it is known that the endothelial-like cells (EL) ori- gin from cancer cells are different from the endothelial cells. However, the detailed difference and the mechan- isms are not well understood. In the present study, we set out to determine some biological behaviors of the ELs from two malignant ovarian cancer cell lines, SKOV-3 and ES-2, such as the proliferation, cell cycle, apoptosis, the activity of telomerase and invasion. At the same time, we compared these biological behaviors with traditional endothelial cell, human umbilical vein endothelial 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 endothe- lial cells (HUVEC) were isolated from umbilical vein and cultured as described previously [14] 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, fol- lowed by 30 min’s incubation at 37°C in a humidified 5% CO2 incubator, as described previously [15]. 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 [16]. The cells were treated with 50 nM Sirolimus (Sigma, St. Louis, MO) in DMSO to inhibit the role of HIF-1a 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 nor- moxia 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 tryp- sinized 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 cen- trifuged, washed and resuspended in 500 μl PBS con- tained 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. At least 20000 cells were analyzed per sample. Cell proliferation characters were indexed by the ratio in S-phase. Invasion assay Invasion assays were performed in a 24-well transwell chamber (Costar, Bodenheim, Germany) as previously described [17]. Briefly, the 8 μm pore inserts were coated with 15 μg of Matrigel. Cells were seeded to coated filters (5 × 104 cells/filter) in 200 μL of serum- free medium in triplicate. Another 500 μL of serum-free media was added in the lower parts of the chambers. After 7d’s incubation under hypoxia, the upper Matrigel coated surface was wiped off using a cotton swab. Cells