Everolimus

Administered via p.o.

BT474 cell line and the primary breast cancer cells

Everolimus is currently in Phase I clinical trials in patients with Unspecified Adult Solid Tumor.

Everolimus (RAD001) is an mTOR inhibitor of FKBP12 with IC50 of 1.6-2.4 nM.

Everolimus is dissolved in saline.

Everolimus exhibits the immunosuppressive activity which is comparable to that of rapamycin. Everolimus competes with immobilized FK 506 for binding to biotinylated FKBP12 and shows the inhibitory effect on a two-way MLR performed with spleen cells from BALB/c and CBA mice with IC50 of 0.12-1.8 nM. [1] Everolimus also shows antiangiogenic/vascular effects in VEGF-induced HUVEC proliferationwith IC50 of 0.12 nM and bFGF-induced HUVEC proliferation with IC50 of 0.8 nM, respectively. [2] A recent study shows that Everolimus shows a dose-dependent inhibitory effects on both the total cells and the stem cells from the BT474 cell line and the primary breast cancer cells with IC50 of 156 nM in total cells of primary breast cancer cells and 71 nM in total cells of BT474 cells. In addition, combination treatment with Everolimus and trastuzumab produces the significantly increased inhibition on the growth of cancer stem cells with the inhibition rate increased by more than 50 %. [3]

24 hours

The 3-(4-5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide dye reduction assay (MTT assay) is used to compare the effects of Everolimus or trastuzumab on total breast cancer cells and breast CSCs. The total cells and the stem cells from the BT474 cell line and the primary breast cancer cells are respectively seeded into 96-well plates with different concentrations of the drugs, with five wells for each concentration, and the cells are cultured at 37 C with 5 % CO2 in an incubator for 24 hours. The concentrations of Everolimus are 1 nM, 10 nM, 100 nM, 1 uM and 10 uM, and the concentrations of trastuzumab are 0.5 ug/mL, 1 ug/mL, 10 ug/mL, 50 ug/mL, and 100 ug/mL. The combinatorial inhibitory effect of Everolimus and Trastuzumab on the in vitro growth of breast CSCs is examined by MTT assay as well using 10 ug/mL trastuzumab in combination of increasing concentrations of everolimus (1 nM, 10 nM, 100 nM and 1 uM). After drug treatment for 24 hours, 20 uL MTT [5 mg/mL in phosphate buffered saline (PBS)] is added to each well, and the cells are incubated at 37 C with 5 % CO2 and saturated humidity for 4 hours. Following the subsequent removal of the supernatant, 150 uL dimethyl sulfoxide (DMSO) is added to each well, and the cells are vortexed for 10 minutes. The light absorbance (OD value) is measured for each well using an ELISA reader. Each experiment is repeated in triplicate, and dose–response curves are plotted. The probit software of the statistical software package SPSS 17.0 for Windows is used to calculate the inhibitory concentration (IC50) of Everolimus.

Everolimus (RAD001) Chemical Structure

DMSO 30 mg/mL, Water <1 mg/mL, Ethanol 7 mg/mL