Effects of Hsp90 inhibitors, geldanamycin and its analog, on ceramide metabolism and cytotoxicity in PC12 cells.

The inhibitors of heat shock protein-90 (Hsp90), geldanamycin (GA) and 17-(allylamino)-17-desmethoxygeldanamycin, show various cellular effects including destabilization of Hsp90 clients and expression of other chaperones, etc. and modulate cytotoxicity depending on cell types and stimuli. In this study, we investigated the effects of Hsp90 inhibitors on survival of PC12 cells with and without cytotoxic stimuli including orthovanadate, Na(3)VO(4). Treatment with Hsp90 inhibitors at 2 µM for 16 hr did not cause cell detachment and leakage of lactate dehydrogenase, and at concentrations greater than 5 µM resulted in cytotoxicity. The inhibitors at 2 µM enhanced the cytotoxicity of 1 mM Na(3)VO(4), and did not protect PC12 cells at any concentrations against Na(3)VO(4). Next, the effects of Hsp90 inhibitors on the intracellular metabolism of ceramide and arachidonic acid (AA) were examined, since these processes also regulate cytotoxicity. In cells treated with 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled C6-ceramide, Hsp90 inhibitors reduced the formation of NBD-glucosylceramide and Na(3)VO(4)-induced formation of NBD-caproic acid, a counterpart of sphingosine, without affecting other metabolites including NBD-sphingomyelin. GA treatment did not change the amounts of AA released in PC12 cells with and without Na(3)VO(4). In HeLa cells, however, GA treatment decreased the release of AA via cytosolic phospholipase A(2)α's activation probably because of dysfunctional Hsp90 clients. Our results suggest the possible involvement of ceramide metabolism, not AA release, in GA-induced cytotoxicity in PC12 cells.