Fig. 7. Effect of DHA on proliferation, invasion and P-gp expression on doxorubicin resistant MCF-7 breast cancer cell.

For cell proliferation assay MCF-7dox cells were plated in a 96 well plate. After 24 hours of incubation, cells were replenished with fresh media with different concentrations of DHA and doxorubicin (2μM) alone or in combination. Cells were then incubated for an additional 48 hours. At the end of incubation, 20 μl MTS reagent was added to each well, and incubated for 4 hours at 37C. Absorbance was read at 490nm. For invasion assay, MCF-7dox cells in 200 μl serum free DMEM was added to upper chamber and 700 μl of DMEM supplemented with 10% fetal calf serum (FCS) and DHA (50 μM) and doxorubicin (2 μM) alone or in combination added to the lower chamber in a 24-well BioCoat Matrigel invasion coated chamber inserts with 8-μm pore size membranes. After 48 hours of incubation, the remaining upper chamber cells were removed and cells which had migrated through the pores to the lower side of the membrane were fixed with 10% formalin and stained with 0.1% crystal violet blue and counted manually. P-gp protein levels were analyzed in MCF-7dox cells treated with 50 μM of DHA and 2 μM of doxorubicin alone or in combination for 48 hours by western blot. Each value represents the mean ± SEM of two independent triplicate cultures. * p<0.05 vs. DHA or doxorubicin alone; ** p<0.05 vs. control or Doxorubicin alone; # p<0.05 vs. doxorubicin alone; ## p<0.05 vs. control or Doxorubicin alone.