Our results Fosbretabulin suggest that HA117 is a strong MDR gene and that its MDR index is similar to that of MDR1 for P-gp substrate drugs and much higher than that of MDR1 for P-gp non-substrate drugs. In addition, using the breast cancer cell line, we show that the MDR mechanism of HA117 may not be similar to that of MDR1. As such, further studies need to be conducted to determine the mechanism of

HA117 to promote MDR. Materials and methods Cell culture The HEK 293 cell line was a generous gift from professor Tong-Chuan He (Selleckchem CP-690550 laboratory of Molecular Oncology, University of Chicago, USA). The breast cancer cell line 4T1 was bought (ATCC, USA) and preserved in our laboratory. The cells were maintained in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and RPMI-1640 medium

(Gibco, USA) supplemented with 10% FBS (Gibco, USA), respectively at 37°C in a humidified atmosphere of 5% CO2. The cells were passaged approximately once every 3 days. Preparation of high titer adenovirus vector supernatant Recombinant adenoviral vectors expressing green fluorescence protein (GFP) and HA117 (Ad-GFP-HA117), GFP and MDR1 (Ad-GFP-MDR1) or only GFP (Ad-GFP) were previously constructed in our laboratory [10]. HEK 293 cells were transducted with Ad-GFP-HA117, Ad-GFP-MDR1 or Ad-GFP viral supernatant at a multiplicity of infection (MOI) CP673451 of 2-5. When all the cells exhibited a round morphology and approximately 80% of them were detached from the culture flask (usually 4 to 5 d post-transduction), the cells were harvested and combined. The cells were then frozen using a dry ice/methanol bath, immediately thawed in a 37°C water bath, and vortexed. A total of 4 freeze/thaw/vortex cycles were performed. After expanding for 3 cycles and purifying using density gradient centrifugation, the high titer recombinant

adenoviruses Ad-GFP-HA117, Ad-GFP-MDR1 selleck chemicals llc and Ad-GFP were harvested, filtered in a aseptic conditions through a 0.45-μm filter and stored at -80°C [11]. Transduction of 4T1 cells with adenoviral vector supernatant Logarithmic phase 4T1 cells were divided into 4 groups. Cells in group 1 were transducted with Ad-GFP-HA117 and cells in group 2 were transducted with Ad-GFP-MDR1 and served as the experimental groups. the stable transductants of these cells in the two groups are referred to as 4T1/HA117 and 4T1/MDR1. A third group of cells was transducted with empty Ad-GFP and served as a control group. the stable transductants of these cells are referred to as 4T1/GFP. Untransducted cells served as a blank control and are referred to as 4T1. The cells were plated on 96-well plates at a density of 2.0 × 105 cells/well and incubated for 16 h.