Abstract
Heat shock protein 90 (HSP90) is a molecular chaperone that regulates the functional stability of client oncoproteins, which is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, 17-AAG also induces transcription of antiapoptotic HSP70 and HSP27 resulting in cell death resistance. The nucleoside analog 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trial, is a transcription inhibitor that gets metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Based on its mechanisms of action we hypothesize that the combination of 17-AAG and 8-Cl-Ado will result in a favorable effect on cytotoxicity.
| . | . | . | % Cell Death . | ||
|---|---|---|---|---|---|
| . | . | . | Annexin V / 7-AAD . | ||
| . | Treatments . | Hrs . | MM.1S . | 8226-RPMI . | U266 . |
| 1) | 17-AAG 0.5 µM | 8 | 5 | 4 | 5 |
| 2) | 17-AAG 0.5 µM | 20 | 2 | 4 | 6 |
| 3) | 8-Cl-Ado 10 µM | 12 | 5 | 3 | 2 |
| 4) | 8-Cl-Ado 10 µM | 20 | 22 | 8 | 2 |
| 5) | 17-AAG→8-Cl-Ado | 8→12 = 20 | 15 | 11 | 10 |
| 6) | 8-Cl-Ado→17-AAG | 12→8 = 20 | 55 | 15 | 19 |
| 7) | 8-Cl-Ado+17-AAG | 20 | 42 | 14 | 34 |
| . | . | . | % Cell Death . | ||
|---|---|---|---|---|---|
| . | . | . | Annexin V / 7-AAD . | ||
| . | Treatments . | Hrs . | MM.1S . | 8226-RPMI . | U266 . |
| 1) | 17-AAG 0.5 µM | 8 | 5 | 4 | 5 |
| 2) | 17-AAG 0.5 µM | 20 | 2 | 4 | 6 |
| 3) | 8-Cl-Ado 10 µM | 12 | 5 | 3 | 2 |
| 4) | 8-Cl-Ado 10 µM | 20 | 22 | 8 | 2 |
| 5) | 17-AAG→8-Cl-Ado | 8→12 = 20 | 15 | 11 | 10 |
| 6) | 8-Cl-Ado→17-AAG | 12→8 = 20 | 55 | 15 | 19 |
| 7) | 8-Cl-Ado+17-AAG | 20 | 42 | 14 | 34 |
As shown in the Table, simultaneous or sequential combination (rows 6 and 7) were more than additive in multiple myeloma cell lines. With 17-AAG alone, there was a transcriptional induction of HSP90α, HSP90β, HSP70, HSC70 and HSP27 as measured by real time RT-PCR. With the drug combinations, there was no reduction of the 17-AAG-induced HSPs mRNA levels, rather a several fold increase was observed for the stress-inducible HSP70 and HSP27. In accordance, the protein levels of the HSPs were also increased when treated with 17-AAG but remained the same when 8-Cl-Ado was added. This suggests that the transcription inhibition by 8-Cl-Ado was not able to abrogate 17-AAG-mediated HSP induction. The transcript levels for client proteins (STAT3, Akt and c-Raf) remained at endogenous levels when treated with the drugs alone or in combination. However, the combination treatments resulted in a greater decline of the protein expression of STAT3 (1-60%), Akt (42-78%), and Raf-1 (25-85%). This may be due to the reduction in intracellular ATP levels after 8-Cl-Ado treatment, MM.1S (57%), 8226-RPMI (24%), and U266 (56%). Taken together, the increased cytotoxicity of 17-AAG and 8-Cl-Ado combination is mediated by the actions of 8-Cl-Ado on the cellular ATP pool and not due to its effect on transcription.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2633.
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