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Temozolomide down-regulates P-glycoprotein in human blood–brain barrier cells by disrupting Wnt3 signaling

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Abstract

Low delivery of many anticancer drugs across the blood–brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/β-catenin signaling, and reduces the binding of β-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.

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Abbreviations

GBM:

Glioblastoma multiforme

CNS:

Central nervous system

BBB:

Blood–brain barrier

TMZ:

Temozolomide

ABC:

ATP-binding cassette

BAT:

Brain-adjacent to tumor

Pgp:

P-glycoprotein

MRP:

Multidrug resistance-related protein

BCRP:

Breast cancer resistance protein

LRP:

Low-density lipoprotein receptor-related protein

GSK3:

Glycogen synthase kinase 3

TCF/lEF:

T cell factor/lymphoid enhancer factor

Dkk-1:

Dickkopf-1

HBMECs:

Human brain microvascular endothelial cells

FCS:

Fetal calf serum

BSA:

Bovine serum albumin

FITC:

Fluorescein isothiocyanate

ZO-1:

Zonula occludens-1

TBP:

TATA-box binding protein

qRT-PCR:

Quantitative real-time PCR

ChIP:

Chromatin immunoprecipitation

MSP:

Methylation-specific PCR

DAPI:

4′,6-Diamidino-2-phenylindole dihydrochloride

LDH:

Lactate dehydrogenase

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Acknowledgments

We are indebted to Prof. Michele Lanotte (Department of Neuroscience, Neurosurgical Unit, University of Turin) and to Dr. Rossella Galli (San Raffaele Scientific Institute, Milan) for providing the primary glioblastoma samples. We are grateful to Costanzo Costamagna (Department of Oncology, University of Turin) for the technical assistance and to Dr. Oriana Monzeglio (Neuro-bio-oncology Center, Policlinico di Monza Foundation) for the technical suggestions with methylation promoter assay. This work has been supported by grants from Compagnia di San Paolo, Italy (Neuroscience Program; grant 2008.1136) and Italian Association for Cancer Research (AIRC; MFAG 11475) to Chiara Riganti. Martha Leonor Pinzón-Daza is recipient of a ERACOL Erasmus Mundus fellowship. Joanna Kopecka is recipient of a “Mario and Valeria Rindi” fellowship provided by Italian Foundation for Cancer Research (FIRC).

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None.

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Authors and Affiliations

  1. Department of Oncology, University of Turin, Via Santena, 5/bis, 10126, Turin, Italy

    Chiara Riganti, Iris C. Salaroglio, Martha L. Pinzòn-Daza, Ivana Campia, Joanna Kopecka, Amalia Bosia & Dario Ghigo

  2. Research Center On Experimental Medicine (CeRMS), University of Turin, Via Santena, 5/bis, 10126, Turin, Italy

    Chiara Riganti, Amalia Bosia & Dario Ghigo

  3. Unidad de Bioquímica, Facultad de Ciencias Naturales y Matemáticas, Universidad del Rosario, Carrera 6, Bogotá, Colombia

    Martha L. Pinzòn-Daza

  4. Neuro-bio-oncology Center, Policlinico di Monza Foundation, Via Pietro Micca 29, 13100, Vercelli, Italy

    Valentina Caldera, Marta Mellai, Laura Annovazzi & Davide Schiffer

  5. Institut Cochin, Centre National de la Recherche Scientifique UMR 8104, Institut National de la Santé et de la Recherche Médicale (INSERM) U567, Université René Descartes, 22 rue Méchain, 75014, Paris, France

    Pierre-Olivier Couraud

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  1. Chiara Riganti
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Correspondence to Chiara Riganti.

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Riganti, C., Salaroglio, I.C., Pinzòn-Daza, M.L. et al. Temozolomide down-regulates P-glycoprotein in human blood–brain barrier cells by disrupting Wnt3 signaling. Cell. Mol. Life Sci. 71, 499–516 (2014). https://doi.org/10.1007/s00018-013-1397-y

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