Synergistic Effect Of Nifurtimox and Inhibition Of Hsp70/Hsp90 In Treatment Of Neuroblastoma
Authors: Rohrer Karin Melanie , Bruchelt Gernot , Handgretinger Rupert , Holzer Ursula 
Published: October 2019

Abstract
Neuroblastoma is the most common solid cancer in childhood with high relapse and mortality rates. Furthermore, high risk neuroblastoma is often accompanied by an infaust prognosis. The 5-nitrofuran nifurtimox, usually used in the treatment of Chagas disease, showed cytotoxic effects against neuroblastoma in vitro and in experimental therapy, which is presumably due to the formation of oxidative stress. Inducing oxidative stress is a well investigated and suitable strategy in the treatment of malignant diseases in vitro but often encounters difficulties in clinical administration. Thus, nifurtimox as a well-established drug represents a promising new approach in treating neuroblastoma. Combining the induction of reactive oxygen species by application of nifurtimox with a blockade of the cells’ own stress response might even increase the cytotoxic effects. The chaperones heat shock protein 70 and 90 (Hsp70/Hsp90) are responsible for refolding or degrading damaged proteins, especially after stress situations such as heat or oxidative stress. Therefore, the roles of Hsp70 and Hsp90 were investigated in more detail.
The commercially available human neuroblastoma cell lines IMR-32, LA-N-1 and the cell line LS, which has been established in the children’s hospital Tuebingen, were exposed to increasing doses of nifurtimox (0.070 mM to 0.348 mM) and incubated for 1, 2 or 3 days. It could be observed that cell viability of all cell lines was significantly and dose-dependently reduced (p<0.01) after nifurtimox treatment. An average reduction of cell viability by 50% was achieved after 24h incubation with 0.348 mM nifurtimox (LS and IMR-32). The assumption that nifurtimox induces the formation of reactive oxygen species could be confirmed. The amount of intracellular reactive oxygen species was significantly increased (p<0.05) in a dose-dependent manner in all cell lines after 24h. Furthermore, expression levels of heat shock proteins Hsp70 and Hsp90 were investigated. Western blot analysis revealed increased intracellular expression levels for both heat shock proteins after 24h nifurtimox treatment. Concluding that Hsp70 and Hsp90 have important roles in tumor cell survival, it was decided to specifically inhibit Hsp90. For this purpose, the neuroblastoma cell lines were treated with the geldanamycin analog 17-Dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). After inhibition of Hsp90 cells were additionally incubated with the previously used dosages of nifurtimox. A significant higher reduction of the cell viability (p<0.001) could be observed for all neuroblastoma cell lines compared to the application of nifurtimox or 17-DMAG alone.
In conclusion, nifurtimox increases oxidative stress in neuroblastoma cell lines leading to significantly decreased cell viability. The specific inhibition of Hsp90 additionally intensifies this effect. The findings suggest that the combined administration of nifurtimox and the specific Hsp90 inhibitor 17-DMAG leads to a synergistic and favorable effect in the treatment of neuroblastoma. More importantly, being an approved medication and well investigated in a wide variety of clinical trials, nifurtimox and 17-DMAG are easy accessible and create a promising new approach not only in neuroblastoma treatment.

Disclosures:
No relevant conflicts of interest to declare.