FK506 Attenuates the MRP1-Mediated Chemoresistant Phenotype in Glioblastoma Stem-Like Cells.

Ángelo Torres,Carlos Oyarzún,José Erices,Valentina Arriagada,Claudia Quezada,Cristian Carrasco,José Rocha,María Toro,Ignacio Niechi

doi:10.3390/ijms19092697

Abstract

Poor response to current treatments for glioblastoma has been attributed to the presence of glioblastoma stem-like cells (GSCs). GSCs are able to expel antitumor drugs to the extracellular medium using the multidrug resistance-associated protein 1 (MRP1) transporter. Tacrolimus (FK506) has been identified as an MRP1 regulator in differentiated glioblastoma (GBM) cells (non-GSCs); however, the effect of FK506 on GSCs is currently unknown. The objective of the following research is to evaluate the effect of FK506 on the MRP1-related chemo-resistant phenotype of GSCs. For this, U87MG and C6 glioma cell lines were used to generate non-GSCs and GSCs. mRNA and MRP1-positive cells were evaluated by RT-qPCR and flow cytometry, respectively. A Carboxyfluorescein Diacetate (CFDA)-retention assay was performed to evaluate the MRP1 activity. Apoptosis and MTT assays were employed to evaluate the cytotoxic effects of FK506 plus Vincristine (MRP1 substrate). GSC-derived subcutaneous tumors were generated to evaluate the in vivo effect of FK506/Vincristine treatment. No differences in transcript levels and positive cells for MRP1 were observed in FK506-treated cells. Lesser cell viability, increased apoptosis, and CFDA-retention in the FK506/Vincristine-treated cells were observed. In vivo, the FK506/Vincristine treatment decreased the tumor size as well as ki67, Glial Fibrillary Acidic Protein (GFAP), and nestin expression. We conclude that FK506 confers a chemo-sensitive phenotype to MRP1-drug substrate in GSCs.

Highlights

Glioblastoma (GBM) is the most common type of brain tumor with the worst prognosis [1,2], which is mainly due to the poor response to the available therapies [3,4,5]
multidrug resistance-associated protein 1 (MRP1) activity decreased up to 33% and 27% in U87MG non-glioblastoma stem-like cells (GSCs) treated with FK506 at concentrations of 7 and 15 ng/mL, respectively (Figure 2A)
C6 GSCs treated with 15 and 30 ng/mL FK506 decreased MRP1 activity in 25% and 33%, respectively (Figure 2B). These results demonstrate that FK506 decreases MRP1 activity in both non-GSCs and GSCs, suggesting that the chemo-sensitizing effect of this drug is due to changes in the activity of the transporter and not its expression

Summary

Introduction

Glioblastoma (GBM) is the most common type of brain tumor with the worst prognosis [1,2], which is mainly due to the poor response to the available therapies [3,4,5]. These treatments involve a trimodal therapy, which consists of surgical resection of the tumor followed by radio- and chemotherapy [5,6]. The aim of this study was to evaluate the effect of FK506 in subtherapeutic concentrations on the chemo-resistant MRP1-associated phenotype in GSCs using in vitro and in vivo assays

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Results

Discussion

Conclusion