Heat shock protein 90 (Hsp90) is a chaperone protein regulating PC-12 cell survival by binding and stabilizing Akt, Raf-1, and Cdc37. Hsp90 inhibitor Geldanamycin (GA) cytotoxicity has been attributed to disruption of Hsp90 binding, and the contribution of oxidative stress generated by its quinone group has not been studied in this context.Reactive oxygen species (ROS) and cell survival were assessed in PC-12 cells exposed to GA or Menadione (MEN), and Akt, Raf-1, and Cdc37 expression and binding to Hsp90 were determined. GA disrupted Hsp90 binding and increased ROS production starting at 1h, and cell death occurred at 6h, inhibited by N-acetyl cysteine (NAC) without preventing dissociation of proteins. At 24h, NAC prevented cytotoxicity and Hsp90 complex disruption. However MnTBAP antioxidant treatment failed to inhibit GA cytotoxicity, suggesting that NAC acts by restoring gluthathione. In contrast, 24h MEN induced cytotoxicity without disrupting Hsp90 binding. GA and MEN decreased Hsp90-binding proteins expression, and proteasomal inhibition prevented MEN, but not GA-induced degradation. In conclusion, while MEN cytotoxicity is mediated by ROS and proteasomal degradation, GA-induced cytotoxicity requires ROS but induces HSP90 complex dissociation and proteasome-independent protein degradation. These differences between MEN and GA-induced cytotoxicity, may allow more specific targeting of cancer cells.

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