Abstract #4050: Increased cytotoxicity with nifurtimox and rapamycin combination therapy in neuroblastoma.

Background: Neuroblastoma is an aggressive solid tumor with poor prognosis, with less than 10% survival in relapsed patients. Novel therapies are needed to help increase the survival and quality of life for these children. We have previously shown that nifurtimox decreases AKT phosphorylation and induces reactive oxygen species (ROS) resulting in apoptosis in neuroblastoma both in vitro and in vivo. Rapamycin is a therapeutic agent potentially effective in neuroblastoma through inhibition of the mTOR regulatory pathway. Targeting the mTOR/AKT pathway at multiple sites, we hypothesize the combination of Nifurtimox with Rapamycin would increase cytotoxicity to neuroblastoma. Methods: Neuroblastoma cell lines (Be(2)C, LA1-55n, SH-SY5Y, SMS-KCNR) were grown in 48-well plates and treated with increasing doses of Nifurtimox (0-20ug/ml) and Rapamycin (0-50nM), separately and in combination for 48 hours. Cell viability was assessed by calcein AM assays. Formation of ROS was examined through DCF absorbance by flow cytometry. Cell lysates were evaluated by western blots for caspase-3. In a xenograft model, 32 nude mice were injected with 10⁷ SMS-KCNR cells subcuetaneously in the left flank. The mice were treated with either control food, or daily doses of either 5 mg/kg Rapamycin, 150 mg/kg Nifurtimox or combination.  Results: The cell viability was decreased in a dose dependent manner with both Nifurtimox and Rapamycin. The combination of Rapamycin and Nifurtimox was more cytotoxic than either drug alone, both in vitro and in vivo. There was a decrease in the IC50 of Nifurtimox in the presence of 25 nM Rapamycin. Cells treated with combined treatment showed an increase in caspase-3 activity confirming apoptosis. An increase in ROS production was detected with DCF flow cytometry. The xenograft model showed a significant decrease in tumor volume in mice treated with both Rapamycin and Nifurtimox when compared to the single agent treatment groups (Control 3.38 cm³, Nifurtimox 2.0 cm³, Rapamycin 1.55 cm³, and Combination 0.73 cm³). Prolonged survival time was also seen in the combination group. Conclusions: Nifurtimox inhibits neuroblastoma growth both in vitro and in vivo when co-administered with Rapamycin to a greater extent than either drug alone. The cytotoxicity involves the production of ROS which induces caspase-3 mediated apoptosis. This combination therapy appears to be effective and well tolerated in the mouse model and would be a novel therapy for neuroblastoma.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 4050.