MYCN and MYC down regulation was clearly BMS-536924 IGF-1R inhibitor observed in IMR5 and SY5Y as early as 3 h of the drug treatment. A small reduction of MYCN and MYC expression was also seen in CHP134 and SKNAS treated with 17 DMAG for 3 and 9 h, respectively. Our previous study indicated that an elevated p53 expression had a suppressive BMS-536924 IGF-1R inhibitor effect on MYCN expression in MYCN amplified neuroblastoma cells. We thus examined if Hsp90 inhibition by 17 DMAG could up regulate p53 expression in neuroblastoma cell lines. The SKNAS cell line was not included in this experiment because it harbors TP53 mutations. As shown in Fig. 3A, treatment of IMR5, CHP134 and SY5Y with 17 DMAG in fact resulted in an increased p53 expression as early as day 1 of the treatment.
Early time course studies showed that the BMS-540215 effect of the drug treatments on p53 expression varied among the cell lines examined.
An enhancement of p53 expression was most apparent in IMR5, in which p53 expression was increased after 6 h of the drug treatment. There was no apparent effect on p53 expression in CHP134 and SY5Y BMS-540215 up to 9 h of the drug treatment. As described, Hsp90 inhibition increased p53 expression in the neuroblastoma cells. We therefore examined if 17 DMAG treatment up regulated the expression of p21WAF1, a known target of p53. As shown in Fig.
4, Hsp90 inhibition by 17 DMAG resulted in an upregulation of p21WAF1 expression in IMR5 and SY5Y cells, but not in CHP134. SKNAS with TP53 mutations showed little induction of p21WAF1 expression upon the drug treatment. AKT is a known client protein of Hsp90, and thus inhibition of Hsp90 leads to degradation of AKT.
In addition, the AKT pathway is known to stabilize MYC and MYCN. We thus examined the effect of Hsp90 inhibition by 17 DMAG on AKT stability in the neuroblastoma cells as a control, and to compare to the MYCN and MYC destabilization described in Fig. 2A. As shown in Fig. 5A, 17 DMAG treatment of the neuroblastoma cells resulted in a decreased AKT expression. Kinetics of AKT destabilization resembled to those of MYCN and MYC down regulation in the neuroblastoma cell lines examined.
In addition, Hsp90 inhibition by 17 DMAG treatments did not change the subcellular localization of AKT, MYCN and MYC in CHP134 and SKNAS cells. Subcellular localization of these proteins in the drug treated IMR5 and SY5Y was not examined.
To address a potential role of Hsp90 inhibition in interfering with mitosis, we examined the expression of acetylated tubulin in the 17 DMAG treated neuroblastoma cells. As shown in Fig. 6, there was an increased expression of acetylated tubulin in the drug treated cells, suggesting that tubulin deacetylase levels were down regulated by Hsp90 inhibition. In fact, expression levels of a tubulin deacetylase, HDAC6, were markedly suppressed in these cells. Favorable neuroblastoma genes are known to be growth suppressive. Since SKNAS is a TP53 mutated cell line, we asked whether Hsp90 inhibition up regulated favorable neuroblastoma genes in SKNAS as an alternative mechanism to p53 pathways in suppressing growth of these cells. As shown in Fig. 7, treatment of SKNAS cells with 17 DMAG resulted in an increased expression of favorable neuroblastoma genes as well as growth suppressive genes . Thus far, MIZ 1 is the only known favorable neuroblastoma g