Inhibition of heat shock protein 90 (Hsp90) sensitizes HER2-negative breast cancer derived MDA-MD-231 cell line to the cytotoxic effect of troglitazone (TRO)

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In this study, we describe how the combination of 50 μM troglitazone (TRO) with 5 μM 17-allylamino-17-demethoxygeldanamycin (17-AAG), shortens the time for the maximum cytotoxic effect to occur from 96 h to 48 h for 17-AAG in HER2-negative breast cancer-derived MDA-MD-231 cell line. The difference in number of viable cells versus controls (26.87 ± 0.88 x 10⁴) in the drug combination (2.87 ± 0.53 x 10⁴) reached statistical significance as compared to each single drug (7.25 ± 0.35 x 10⁴ for TRO, P<0.02, and 12.75 ± 0.35 x 10⁴ for 17-AAG, P<0.01, respectively). Hitherto, we studied whether the increased cytotoxicity observed at 48 h was preceded by an increased cleavage of poly(ADP-ribose) polymerase (PARP) as a result of caspase activation at 24 h. Image software analysis of Western blots expressed as arbitrary units (AU) revealed a 4.8 fold increase of the level of PARP 85 kD fragment in cells treated with 17-AAG/TRO as compared to TRO (AU 8.85 ± 0.67 17-AAG/TRO, and AU 2.07 ± 0.54 TRO, P=0.008). Surprisingly, this difference was mainly due to the action of 17-AAG as revealed by the results of this drug alone at the same time point (AU 8.26 ± 1.68). Furthermore, we investigated whether the level of Akt protein or the level of its phosphorylation, a major target protein of the inhibitory activity of 17-AAG especially in HER2-negative breast carcinoma-derived cells, played any role in the increased cytotoxicity. At 24 h, there was no difference in level of phosphorylation of Akt (p-Akt) by Western blot analyis in any of the cell populations. On the contrary, although the level of Akt decreased in all treated cells as compared to untreated controls, this reached statistical significance only in 17-AAG versus TRO (AU 0.40 ± 0.04 17-AAG, and AU 0.63 TRO, respectively, P=0.018), and in TRO/17-AAG versus 17-AAG (AU 0.27 ± 0.06 TRO/17-AAG, and AU 0.40 ± 0.04 17-AAG, respectively, P=0.016). These data show that Akt-downregulation mediated by inhibition of Hsp90 increases the cytotoxicity of TRO, and support further investigation to understand apoptosis-independent cytotoxic mechanisms solicited by the combination of the drugs in HER2-negative breast cancer cells.