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Open Access Highly Accessed Research article

HIF-1 activation induces doxorubicin resistance in MCF7 3-D spheroids via P-glycoprotein expression: a potential model of the chemo-resistance of invasive micropapillary carcinoma of the breast

Sophie Doublier12*, Dimas C Belisario12, Manuela Polimeni12, Laura Annaratone3, Chiara Riganti12, Elena Allia3, Dario Ghigo12, Amalia Bosia12 and Anna Sapino3

Author Affiliations

1 Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena, 5/bis, 10126 Turin, Italy

2 Center of Experimental Research and Medical Sciences, University of Turin, Turin, Italy

3 Department of Biomedical Sciences and Human Oncology, University of Turin, Turin, Italy

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BMC Cancer 2012, 12:4  doi:10.1186/1471-2407-12-4

Published: 4 January 2012

Abstract

Background

Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal type B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small pseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1 (HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells cultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.

Methods

HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers. Binding of HIF-1α to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by ChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric assay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.

Results

In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of MDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was increased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than MCF7 cells cultured as monolayer. Finally, HIF-1α inhibition either by incubating cells with 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (a widely used HIF-1α inhibitor) or by transfecting cells with specific siRNA for HIF-1α significantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin accumulation in MCF7 3-D spheroids.

Conclusions

MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is associated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same mechanism may be suggested for IMPC drug resistance.

Keywords:
HIF-1α; 3-D spheroids, Elastase; Invasive micropapillary breast carcinoma; Doxorubicin resistance; P-glycoprotein; MUC-1