Geldanamycin decreases Raf‐1 and Akt levels and induces apoptosis in neuroblastomas

Abstract

Neuroblastomas are the most common extracranial solid tumors of childhood. These tumors are associated with an overall poor prognosis, particularly for advanced stage disease. The benzoquinone ansamycin antibiotic, geldanamycin (GA), exhibits potent antitumor activity in certain cancer cell lines by destabilizing important signal transduction proteins (e.g., Raf-1 and Akt). The purpose of our study was to determine whether GA can alter the expression of Raf-1 and Akt, which have been shown to be critical for neuronal cell survival, and induce apoptosis of neuroblastoma cells. Human neuroblastoma cells (SH-SY5Y, SK-N-SH and LAN-1) were treated with GA for a variable period of time. Cell viability was assessed with MTT assays. Apoptosis was assessed with DNA fragmentation ELISA, TUNEL-flow cytometric assay, Western blot and caspase activities. We found that GA decreases cell viability and induces apoptosis in the SH-SY5Y human neuroblastoma cell line. These effects were mediated through activation of caspase-9 and -3, mitochondrial release of cytochrome c and subsequent PARP cleavage. GA-induced apoptosis was associated with a reduction in the level and activity of Raf-1 and Akt. The importance of these proteins was further demonstrated by induction of apoptosis in SH- SY5Y cells by a combination of U0126 (MEK1/2 inhibitor) and LY294002 (an inhibitor of PI3K). Similar to SH-SY5Y cells, other human neuroblastoma cells (SK-N-SH and LAN-1)were sensitive to the effects of GA-induced apoptosis. Taken together, our findings suggest that GA may be a novel therapeutic agent, which may be effective in the treatment of neuroblastomas.

Hsp90 inhibitors deplete key anti‐apoptotic proteins in pediatric solid tumor cells and demonstrate synergistic anticancer activity with cisplatin

“…Because of its role in anti-apoptotic signaling, the Hsp90 client protein AKT was also selected for study as a potential marker of the activity of Hsp90 inhibitors in pediatric tumor cells. We were able to show marked decreases in levels of these proteins after GA exposure in all of the cell lines evaluated, and our findings are in agreement with those reported by Kim et al 50 regarding modulation of phospho-AKT levels in neuroblastoma cells after exposure to GA.…”

“…exposed to GA as a single agent, the IC 50 was 2.4 M. Because the ratio of GA to cisplatin was fixed at 1:100 when the drugs were used in combination, the GA concentration at the IC 50 for GA ϩ cisplatin was only 0.039 M. The addition of cisplatin thus resulted in a marked decrease in the IC 50 of GA in SAOS cells. Median dose analysis indicated that the combination of GA and cisplatin resulted in greater-than-additive inhibition of cell survival/proliferation as shown in Figure 4b.…”

Expression of heat shock protein 90 at the cell surface in human neuroblastoma cells

“…Hsp90 inhibitor 17-AAG was a potent cytotoxic agent for human neuroblastoma NB69 cells, inducing cell death at concentrations ranging from 10 to 60 nM. These inhibitor concentrations were in the described range for SH-SY5Y cells (Kim et al 2003) and other tumor cell types with higher binding affinity to 17-AAG (Kamal et al 2003). Interestingly, proliferative spherical cells were significantly more sensitive to 17-AAG compared to differentiated flattened cells.…”

“…The figure is a representative result. The positions of relative molecular weight standards (in thousands) are indicated described as potent anti-tumor agents in certain tumor cells (Kamal et al 2003;Zhang et al 2007), including some human neuroblastoma cell lines (Kim et al 2003;Kang et al 2006), but the neuroblastoma NB69 cell line has not yet been studied. Hsp90 inhibitor 17-AAG was a potent cytotoxic agent for human neuroblastoma NB69 cells, inducing cell death at concentrations ranging from 10 to 60 nM.…”

“…This result was coincident with the increased expression of cell-surface Hsp90 in these spherical neuroblastoma cells. It has been reported that geldanamycin induces apoptosis in human neuroblastoma cells SH-SY5Y, LAN-1, and SK-N-SH (Kim et al 2003;Shen et al 2007). Hsp90 inhibitor 17-AAG, at the described nanomolar concentrations, also induced cell death by apoptosis in cultured neuroblastoma NB69 cells (not shown).…”