| In Vitro | In vitro activity: 17-AAG, an analog of geldanamycin, exhibits greater than 100 times higher binding affinity for Hsp90 derived from HER-2-overexpressing cancer cells (BT474, N87, SKOV3 and SKBR3) or BT474 breast carcinoma cells with IC50 values of 5-6 NM. 17-AAG causes the degradation of HER2, HER3, Akt, and both mutant and wild-type androgen receptor (AR), leading to the RB-dependent G1 growth arrest of prostate cancer cells such as LNCaP, LAPC-4, DU-145, and PC-3 with IC50 values of 25-45 NM. In addition to inducing apoptosis of Ba/F3 cells transformed with wild-type BCR-ABL with an IC50 of 5.2 μM, 17-AAG has the ability to induce apoptosis of cells transformed with imatinib mesylate-resistant T315I and E255K BCR-ABL mutants with IC50 values of 2.3 μM and 1.0 μM, respectively, by inducing the degradation of both wild-type BCR-ABL protein and mutants.
Kinase Assay: Purified native Hsp90 protein or cell lysates from HER-2-overexpressing cancer cells (BT474, N87, SKOV3 and SKBR3) or BT474 breast carcinoma cells in lysis buffer (20 mM HEPES, pH 7.3, 1 mM EDTA, 5 mM MgCl2, 100 mM KCl) are incubated with various concentrations of 17-AAG for 30 minutes at 4 °C, and then incubated with biotin-GM linked to BioMag streptavidin magnetic beads for 1 hour at 4 °C. Tubes are placed on a magnetic rack, and the unbound supernatant removed. The magnetic beads are washed three times in lysis buffer and heated for 5 minutes at 95 °C in SDS–PAGE sample buffer. Samples are analysed on SDS protein gels, and western blots done using indicated antibodies. Bands in the western blots are quantified using the Bio-rad Fluor-S MultiImager, and the percentage inhibition of binding of Hsp90 to the biotin-GM is calculated. The IC50 reported is the concentration of 17-AAG needed to cause half-maximal inhibition of binding.
Cell Assay: Cells (BT474, SKBR3, N87, SKOV3, MCF7, MDA468, Hs578T, Hs578Bst, A549, HT29, U87, SKMG3, HT1080, RPTEC, NDF, HMVEC, HMEC, HUVEC, and PBMC cells) are seeded in 96-well plates at 2,000 cells per well in a final culture volume of 100 μL for 24 hours before the addition of increasing concentrations of 17-AAG that is incubated for 5 days. Viable cell number is determined using the Celltiter 96 AQueous Nonradioactive Cell Proliferation Assay. The value of the background absorbance at 490 nm (A490) of wells not containing cells is subtracted. Percentage of viable cells = (A490of 17-AAG treated sample/A490 untreated cells) × 100. The IC50 is defined as the concentration that gave rise to 50% viable cell number. |
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