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.
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.
Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena, 5/bis, 10126 Turin, Italy. sophie.doublier@unito.it
Sophie
S
Doublier
Dimas C
DC
Belisario
Manuela
M
Polimeni
Laura
L
Annaratone
Chiara
C
Riganti
Elena
E
Allia
Dario
D
Ghigo
Amalia
A
Bosia
Anna
A
Sapino
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D017302:Annexins / Q000032:analysis; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000188:drug therapy / Q000378:metabolism; D002291:Carcinoma, Papillary / Q000188:drug therapy / Q000378:metabolism; D020169:Caspases / Q000032:analysis; D004317:Doxorubicin / Q000494:pharmacology*; D019008:Drug Resistance, Neoplasm / Q000502:physiology*; D005260:Female; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000378:metabolism*; D018874:Spheroids, Cellular; D014407:Tumor Cells, Cultured
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D017302:Annexins; D000970:Antineoplastic Agents; D051793:Hypoxia-Inducible Factor 1; D004317:Doxorubicin; D020169:Caspases