Nifurtimox reduces N-Myc expression and aerobic glycolysis in neuroblastoma

Abstract

Neuroblastoma is one of the most common solid tumors in childhood and usually accompanied with poor prognosis and rapid tumor progression when diagnosed with amplification of the proto-oncogene N-Myc. The amplification of N-Myc has major influence on the maintenance of aerobic glycolysis, also known as the Warburg effect. This specific switch in the conversion of pyruvate to lactate instead of the conversion of pyruvate to acetyl-coenzyme A even in the presence of oxygen has important benefits for the tumor, e.g. increased production of enzymes and enzyme substrates that are involved in tumor progression, angiogenesis and inhibition of apoptosis. The antiprotozoal drug nifurtimox, which is generally used for the treatment of infections with the parasitic protozoan Trypanosoma cruzi, has been reported to have cytotoxic properties in the therapy of neuroblastoma. However, its action of mechanism has not been described in detail yet. The presented in vitro study on the neuroblastoma cell lines LA-N-1, IMR-32, LS and SK-N-SH shows an increased production of oxidative stress, a reduced lactate dehydrogenase enzyme activity and reduced lactate production after nifurtimox treatment. Furthermore, nifurtimox leads to reduced mRNA and protein levels of the proto-oncogene protein N-Myc. Thus, the current work gives new insights into the effect of nifurtimox on tumor metabolism revealing a shifted glucose metabolism from production of lactate to oxidative phosphorylation and a reduced expression of the major molecular prognostic factor in neuroblastoma N-Myc, presenting nifurtimox as a possible adjuvant therapeutic agent against (high risk) neuroblastoma.

Keywords: N-Myc; Warburg effect; aerobic glycolysis; neuroblastoma; nifurtimox.

Figure 1.

For figure legend, see page .

Figure 2.

Cell viability after nifurtimox and/or topotecan treatment. (A) Data show cell viability of cell line LS (N-Myc amplified) in reference to vehicle control after 48h incubation with topotecan as indicated, growth medium alone (untreated) or vehicle control tested with a MTS cell viability assay. Sample size n = 4. Data show mean ± standard deviation. Significant changes vs. vehicle control tested via ANOVA indicate P < 0.0001 (****). (B) Data show cell viability of cell line LS (N-Myc amplified) in reference to vehicle control after 48h incubation with nifurtimox as indicated or nifurtimox + 0.2 µM topotecan tested with a MTS cell viability assay. Sample size n = 4. Dotted line indicates cell viability after 48h incubation with 0.2 µM topotecan alone. Significant differences were tested with an ANOVA and Sidak-Bonferroni correction for multiple comparison (****: P < 0.0001); “a” indicates significant difference to treatment group 0.2 µM topotecan alone.

Figure 3.

Expression of N-Myc. (A). Data show mRNA expression levels in reference to vehicle control after 24h incubation with nifurtimox as indicated measured with quantitative real time PCR. Sample size n = 3. (B) Data show absolute Cq-values of the data in Fig. 3A to illustrate absolute N-Myc mRNA Expression levels using an N-Myc amplified cell line IMR-32 and the non-amplified cell line SK-N-SH. Sample size n = 3. (C) Data show protein expression levels of N-Myc in reference to vehicle control after 4 h incubation with nifurtimox as indicated measured with quantitative western blot (cell line LA-N-1). Data show mean ± standard deviation. Significant changes versus untreated/DMSO tested via ANOVA (A) or unpaired Student's t-test (B) indicate P <0.001 (***).

Figure 4.

Glycolysis. (A) Data show consumption of β-D-(+)-glucose per 4 h in the supernatant growth medium after incubation with nifurtimox or growth medium alone normalized to the number of cells. Consumption of D-Glucose was significantly reduced for LA-N-1 (P = 0.0101), IMR-32 (P = 0.0057), LS (P = 0.0069) and SK-N-SH (P = 0.0078) after treatment with nifurtimox. Experiments were carried out in duplicates and represent one of 3 independent experiments with similar outcome. (B) Data show production of L-lactate per 4 h in the supernatant growth medium after incubation with nifurtimox or growth medium alone normalized to the number of cells. Production of lactate was significantly reduced for LA-N-1 (P = 0.0103), IMR-32 (P = 0.0214), LS (P = 0.0242) but not for SK-N-SH (P = 0.2753) after treatment with nifurtimox. Experiments were carried out in duplicates and represent one of 3 independent experiments with similar outcome. Data show mean ± standard error of the means. Significant changes vs. untreated tested via ANOVA indicate P < 0.05 (*) and P < 0.01 (**).

Figure 5.

Reduction of phospho-PDH levels after treatment with nifurtimox. (A) Data show protein levels of pyruvate dehydrogenase (PDH) and S293-phosphorylated pyruvate dehydrogenase (phospho-PDH) in reference to control after 4h incubation with nifurtimox as indicated or growth medium alone measured with quantitative western blot. Sample size n = 3. Data show mean ± standard deviation. Significant changes versus untreated control tested via unpaired Student's t-test indicate P < 0.01 (**), P < 0.001 (***) and P < 0.0001 (****). (B) Example blots of each cell line; protein bands are combined accordingly.

Figure 6.

Enzyme activity of lactate dehydrogenase after nifurtimox treatment. Data show lactate dehydrogenase enzyme activity after 4 h incubation with nifurtimox as indicated or growth medium alone. One of 3 independent experiments with similar outcome is shown. Data show mean ± standard error of the means. Significant changes vs. untreated control tested via unpaired Student's t-test indicate P < 0.01 (**) and p < 0.001 (***).