Cell experiment:

The Neuroblastoma cell lines IMR-32, LA-N-1 and SK-N-SH and the neuroblastoma cell line LS are grown in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL Penicillin and 100 μg/mL Streptomycin and incubated at 37℃, 5% CO2 and saturated humidity. To assess the cell viability after incubation with Nifurtimox at different concentrations (10 μg/mL up to 50 μg/mL or 34.8 μM to 174 μM, respectively in the supernatant growth medium) or the vehicle control with according concentrations, all neuroblastoma cell lines are subjected to an MTS assay. Stock solutions of MTS are made at 480 μM in sterile filtered deionized water and stored at -20℃. Cells are grown to approximately 50% confluency, treated with Nifurtimox, and incubated for 1 h with fresh media containing 12 μM MTS[1].

Nifurtimox, an antiprotozoal agent, which is generally used for the treatment of infections with Trypanosoma cruzi, has been used in the therapy of neuroblastoma. Nifurtimox affects enzyme activity of lactate dehydrogenase (LDH).

Nifurtimox affects enzyme activity of lactate dehydrogenase (LDH). To differentiate if this effect is a result of a reduced LDH activity or a shift in pyruvate metabolism due to activation of PDH, the enzyme activity of LDH is determined after 4 h treatment with 50 μg/mL Nifurtimox. Compared to the untreated control, the LDH activity is significantly reduced for LA-N-1 (P=0.005), IMR-32 (P=0.009), LS (P=0.0035) and SK-N-SH (P=0.0065). Nifurtimox reduces cell viability and induces cell cycle arrest and apoptosis in neuroblastoma cells. To characterize the cytotoxic impacts of Nifurtimox on neuroblastoma, 4 cell lines are subjected to several experiments. Cell viability is reduced for all 4 neuroblastoma cell lines after 24 h incubation with 50 μg/mL to an average of 66%, 63%, 62% and 75% (LA-N-1, IMR-32 LS and SK-N-SH, respectively). The reduction is significant compared to the untreated control (P<0.01) and the vehicle control with DMSO (P<0.05) for all cell lines[1].

References:
[1]. Cabanillas Stanchi KM, et al. Nifurtimox reduces N-Myc expression and aerobic glycolysis in neuroblastoma. Cancer Biol Ther. 2015;16(9):1353-63.