Nifurtimox Inhibits the Progression of Neuroblastoma in vivo

Abstract

Neuroblastoma was one of the most life-threatening cancer developed in children, yet the conventional therapies currently used leave an unmet gap for clinical requirements. Temozolomide is the first line of drug in the treatment of neuroblastorma nowadays. Giving the fact that temozolomide treatment offered limited healing effect and patients responded divergently, an alternative beneficial path is urgently requested. Nifurtimox, a drug against Trypanosoma cruzi, was happened to find competent in treating a patient who carried aggressive neuroblastoma. Although in vitro studies demonstrated that nifurtimox has cytotoxic features against tumor cells, a systematic investigation in vivo is generally inadequate. Here we exhibited that nifurtimox could suppress the progression of neuroblastoma in vivo, while maintain the health condition to a great extent. Importantly, as comparing to temozolomide, nifurtimox presented a stronger effect on inhibiting tumor development, strongly suggesting that nifurtimox is a preferential alternative drug in treating neuroblastoma. Additionally, it was shown that Akt-GSK3β signaling cascade was involved in tumor arrest induced by nifurtimox.

Keywords: Neuroblastoma; Nifurtimox; Temozolomide; tumor suppression.

Figure 1

The progression of neuroblastoma is inhibited by Nifurtimox in vivo. A) The expressed in vivo fluorescent signal of SH-SY5Y-luc2-GFP was scanned by IVIS Spectrum CT. The first scan was done after 7 days of tumor cell transplantation and then initiated the drug treatment (Day 1), and two consecutive scans (Day 5 and Day9) were carried out every another 4 days for time series tracking. Different dosage of Temozolomide and Nifurtimox were used for drug treatment groups, control group was treated with the same concentration of DMSO used for dissolving drugs. B) The intensity of the fluorescent signal of SH-SY5Y-luc2-GFP was quantified and compared between different groups. * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 2

Nifurtimox was effective in treating neuroblastoma. A, B, C, D, E) representative images of treated mouse (Control and treated groups) were scanned either from overview or side-view, the fluorescence signal of tumor cells in situ was magnified and illustrated for comparison.

Figure 3

Nifurtimox impeded the expansion of neuroblastoma in situ. A) both kidneys were dissected out for direct evaluation of tumor progression. B, C, D) absolute or relative kidney weight was quantified between different treating groups for comparison. Nifurtimox was used for different concentration (50, 100 and 200 mg/kg), Temozolomide (30mg/kg) was used as positive control while DMSO treated group was used as control. * p≤0.05, ** p≤0.01, *** p≤0.001.

Figure 4

The body constitution is well maintained for Nifurtimox treatment. A and B) The absolute or relative body weight of mice were measured daily and plotted together for comparison. Nifurtimox was used for different concentration (50, 100 and 200 mg/kg), temozolomide (30mg/kg) was used as positive control while DMSO treated group was used as control. * p≤0.05 and ** p≤0.01 was labeled as compared with Control group, ^# p≤0.05, ^## p≤0.01, ^### p≤0.001 was labeled as compared with Temozolomide-treated group.

Figure 5

The tumor suppression ratio correlated with the dosage of the Nifurtimox. A and C) the tumor suppression ratio was calculated according to either the tumor-fluorescence intensity or the kidney weight with that of their control. B and D) The obtained tumor suppression ratio correlated perfectly with the dosage of Nifurtimox, R²= 0.9994 or, R²= 1.

Figure 6

AKT-GSK3βsignaling is involved in tumor-suppression induced by nifurtimox. (A-C) Nifurtimox-treated and -untreated SH-SY5Y cells were assayed with Phospho-kinase array, the dot blot was quantified to show that the phosphorylation of GSK3β was significantly upregulated in nifurtimox-treated group. (D) Illustration showed the potential signaling cascade triggered by nifurtimox on the inhibition of tumor growth. (E) The phosphorylation level of Akt and GSK-3β was inhibited by nifurtimox in different cell types e.g. H1299, U251 and N2a. ** p≤0.01 was labeled as compared with Control group.