Table 1.
Analysis of interaction of Hsp90 with LA-12
| Sample | Applied | Result (± SD) | Result |
|---|---|---|---|
| compound | mg Pt/ml | ng Pt/mg protein | |
| D-MEM | negative | - | |
| 10 μM cisplatin | 10 μM cisplatin | 2.09 (0.05) | |
| 50 μM cisplatin | 50 μM cisplatin | 9.93 (0.21) | |
| 10 μM LA-12 | 10 μM LA-12 | 1.96 (0.01) | |
| 50 μM LA-12 | 50 μM LA-12 | 9.14 (0.26) | |
| L1 | negative | - | |
| L2 | 10 μM cisplatin | 0.040 (0.0004) | 20 |
| L3 | 50 μM cisplatin | 0.290 (0.005) | 145 |
| L4 | 10 μM LA-12 | 1.98 (0.02) | 990 |
| L5 | 50 μM LA-12 | 11.6 (0.26) | 5500 |
| P1 | negative | - | |
| P2 | 10 μM cisplatin | - | |
| P3 | 50 μM cisplatin | < 0.02 | |
| P4 | 10 μM LA-12 | 0.034 (0.004) | |
| P5 | 50 μM LA-12 | 0.222 (0.01) | |
| NC1 | 50 μM cisplatin | - | |
| NC2 | 50 μM LA-12 | - | |
| AC1 | 50 μM cisplatin | - | |
| AC2 | 50 μM LA-12 | - | |
Platinum levels were determined by AAS in whole cell lysates (L1-5) of H1299 control cells and cells treated with 10 μM and 50 μM LA-12 or cisplatin for 9 hours. The protein concentration of cell lysates was 2 mg/ml. The amounts of platinum bound to Hsp90 was measured after immunoprecipitation of Hsp90 from H1299 control cells and cells treated with 10 μM and 50 μM LA-12 or cisplatin for 9 hours (P1-5). Immunoprecipitations without antibody (NC1-2) and with DO-1 antibody (AC1-2) in cells treated with 50 μM LA-12 and cisplatin were performed as negative control for crossreactivity of platinum compounds with other reaction components. Control AAS platinum measurements were perfomed in D-MEM cell medium containing 10 μM and 50 μM LA-12 or cisplatin. Results are mean ± SD of triplicate experiments.