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Knockdown of caveolin-1 decreases activity of breast cancer resistance protein (BCRP/ABCG2) and increases chemotherapeutic sensitivity

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Abstract

The ATP-binding cassette transporter breast cancer resistance protein (BCRP/ABCG2) is supposed to be a major determinant of the multidrug resistance phenotype of tumors by extruding chemically diverse cytostatic drugs out of tumor cells. BCRP physically and possibly also functionally interacts with caveolin-1 (CAV1, encoded by Cav1), an integral membrane protein of lipid rafts important for signal transduction and membrane trafficking. Moreover, Cav1 is linked to an aggressive phenotype of cancer cells in various tumors. We therefore investigated whether Cav1 plays a functional role in the regulation of BCRP transport activity and in the resistance against chemotherapeutics that are BCRP substrates. As a cell model, we used the BCRP overexpressing cell line MDCKII-BCRP and the corresponding parental cell line MDCKII as a control. Cav1 expression was down-regulated using retrovirus-mediated RNA interference technology. BCRP activity was assessed by pheophorbide A efflux assay and the resistance towards cytostatic drugs was measured by proliferation assays. Efficient knockdown of Cav1 reduced Cav1 expression by 85–95% and BCRP activity by 35%. Concurrently, it reduced resistance towards the BCRP substrate mitoxantrone but not towards vincristine, a chemotherapeutic that is not extruded by BCRP. Western blot analysis of gradient ultracentrifugation fractions and immunofluorescence demonstrates that BCRP localization within the plasma membrane was largely unaltered in Cav1-deficient cells compared to controls. The diminished BCRP function after Cav1 knockdown is, thus, likely mediated by alterations in protein–protein interactions and suggests a positive regulation of BCRP function by CAV1.

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Abbreviations

ABC:

ATP-binding cassette

AOTF:

Acousto optic tunable filter

BCRP:

Breast cancer resistance protein

Cav:

Caveolin

DMEM:

Dulbecco’s modified Eagle’s medium

DMSO:

Dimethyl sulfoxide

DRM:

Detergent-resistant membrane

EDTA:

Ethylene diamine tetraacetic acid

FTC:

Fumitremorgin C

GFP:

Green fluorescence protein

MDCK:

Madin–Darby canine kidney

MDR:

Multidrug resistance

MF:

Median fluorescence

MRP:

Multidrug resistance-related protein

PBS:

Phosphate-buffered saline

PBS (+):

PBS containing 0.1 mM CaCl2 and 0.1 mM MgCl2

PFA:

Paraformaldehyde

P-gp:

P-glycoprotein

PhA:

Pheophorbide A (3S-(3alpha,4beta,21beta))-14-ethyl-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-9-vinylphorbine-3-propionic acid)

RNAi:

RNA interference

SDS:

Sodium dodecyl sulfate

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

TNE buffer:

TRIS–NaCl–EDTA buffer

TRIS:

2-Amino-2-(hydroxymethyl)-propane-1,3-diol

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Acknowledgments

We thank Dr. A. H. Schinkel (Amsterdam, The Netherlands) for generously providing the cell line MDCKII-BCRP and the National Cancer Institute (Rockville, USA) for providing FTC. Moreover, we would like to thank Richard Sparla for his help with transfection and transduction.

The project was funded by the Wilhelm-Sander Stiftung (#2008.005.1). Caroline Henrike Storch was supported by a fellowship of the Ernst Schering Foundation for doctoral students.

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Authors and Affiliations

  1. Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

    Melanie Herzog, Caroline Henrike Storch, Philipp Gut, Dimitry Kotlyar, Walter Emil Haefeli & Johanna Weiss

  2. Department of Internal Medicine IV, Gastroenterology, Hepatology, Infectious Diseases, and Intoxications, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

    Joachim Füllekrug & Robert Ehehalt

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  1. Melanie Herzog
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  2. Caroline Henrike Storch
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  3. Philipp Gut
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Correspondence to Johanna Weiss.

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Melanie Herzog and Caroline Henrike Storch contributed equally to this work.

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Herzog, M., Storch, C.H., Gut, P. et al. Knockdown of caveolin-1 decreases activity of breast cancer resistance protein (BCRP/ABCG2) and increases chemotherapeutic sensitivity. Naunyn-Schmied Arch Pharmacol 383, 1–11 (2011). https://doi.org/10.1007/s00210-010-0568-8

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