FIGURE 3.

Disruptin inhibits Hsp90 binding to the EGFR and promotes EGFR degradation. A, peptide SVDNPHVC must be internalized to be active at growth inhibition. UMSCC1 cells were grown in 6-well plates to clonal density and treated with 30 μg/ml peptide SVDNPHVC or scrambled peptide with or without mixing with the peptide delivery reagent Chariot. Colonies were counted 8 days after treatment. * denotes significant difference from control at p < 0.05. B, structures of Disruptin and the scrambled peptide. C, effect of Disruptin on Hsp90 interaction with the full-length wild-type EGFR. UMSCC1 or NCI-H1975 cells were treated for 24 h with 50 nm geldanamycin (GA), 30 μg/ml Disruptin, or 30 μg/ml scrambled peptide. Hsp90 was immunoprecipitated (IP), and Hsp90 and EGFR in the immunoprecipitate were detected by immunoblotting. D, time course of Disruptin effect on EGFR levels. UMSCC1 and NCI-H1975 cells treated for 24, 48, or 72 h with 30 μg/ml Disruptin or scrambled peptide were lysed and immunoblotted for EGFR. E, quantitation of Disruptin effect on Hsp90 binding to the EGFR. Hsp90 was immunoprecipitated from UMSCC1 cells treated for 24 h with Disruptin or scrambled peptide as in B. Immunoblots of Hsp90-bound EGFR and total EGFR (Input) were scanned, and the Hsp90-bound EGFR is expressed as % of total receptor. Mean ± S.E. from four experiments. F, disruptin does not inhibit Hsp90 binding to the GR. Aliquots of immunoadsorbed GR were stripped of chaperones with NaCl and incubated with rabbit reticulocyte lysate as described previously (19). After washing, the immune pellets were immunoblotted for GR and associated Hsp90 and Hsp70 or bound with [³H]dexamethasone for assay of steroid binding activity. Lane 1, stripped GR; lanes 2–4, stripped GR incubated with 50 μl of reticulocyte lysate alone (lane 2) or reticulocyte lysate plus 300 μg/ml Disruptin (lane 3) or reticulocyte lysate plus 10 μm geldanamycin (lane 4). G, disruptin does not affect ATP binding activity of Hsp90. UMSCC1 cells were treated for 3 h with 50 nm geldanamycin or 30 μg/ml of Disruptin or scrambled peptide. Cell lysates were prepared and incubated overnight with ATP-agarose beads, and bound Hsp90 was immunoblotted.