Combining tyrosine kinase inhibitors with novel thiosemicarbazone iron chletors in cell lines derived from pediatric solid tumors of neurogenic origin


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Maria Krchniakova1,Jakub Neradil1,Silvia Chovanova1,Petr Chlapek1,Des. R. Richardson2,Jaroslav Sterba3,Renata Veselska1
1Masaryk University,2University of Sydney,3University Hospital Brno

Abstract

Background

Treating pediatric solid tumors is often accompanied with developing multidrug resistance (MDR) that can be caused by drug efflux or lysosomal sequestration. We examined treatment of cell lines derived from neuroblastomas and medulloblastomas with tyrosine kinase inhibitors (TKIs) combined with novel thiosemicarbazones. Thiosemicarbazones are chelators transported into lysosomes where they bind iron and copper ions and create reactive oxygen species that permeate the lysosomal membrane. Previously sequestered drugs are then released, helping overcome MDR.

Method

DAOY (medulloblastoma), SH-SY5Y and SK-N-BE(2) (both neuroblastoma) established cell lines were used. We analyzed phosphorylation of receptor tyrosine kinases (RTKs) using Human Phospho-RTK Array (R&D Systems), and expression of selected ABC transporters by qRT-PCR and Western blot. Selected lysosomotropic TKIs (gefitinib, lapatinib, sunitinib) were combined with thiosemicarbazones (Dp44mT, DpC) and cell viability was measured by MTT assay. The effects of drug combinations were analyzed in CalcuSyn software (Biosoft).

Results

We detected MDR-related transporters: ABCB1 (in SH-SY5Y cells), ABCC1 (in all cell lines) and ABCG2 (in DAOY cells) as well as phosphorylated targets of used TKIs: EGFR (in all cell lines) and PDGFRβ (in neuroblastoma cell lines). Although the selected drugs were effective when used alone, their combined synergistic effects were found only in the DAOY cell line, but not in neuroblastoma cell lines. However, a change in the treatment design resulted in synergy/additivity in these cell lines, except for combinations with lapatinib in the SH-SY5Y cell line. Since lapatinib is an ABCB1 substrate, synergy was achieved after adding an ABCB1 inhibitor Valspodar.

Conclusion

We identified an effective strategy for combining TKIs and thiosemicarbazones in cell lines derived from pediatric medulloblastoma and neuroblastoma, however, our results indicate a different mechanism than we previously hypothesized.

 

Supported by projects no. 17-33104A and 16-34083A (AZV CR), no. LQ1605 (MEYS CR, NPU II) and Brno Ph.D. Talent.