Background: Invasive micropapillary carcinoma (IMPC) of the breast is a distinct and aggressive variant of luminal\r\ntype B breast cancer that does not respond to neoadjuvant chemotherapy. It is characterized by small\r\npseudopapillary clusters of cancer cells with inverted cell polarity. To investigate whether hypoxia-inducible factor-1\r\n(HIF-1) activation may be related to the drug resistance described in this tumor, we used MCF7 cancer cells\r\ncultured as 3-D spheroids, which morphologically simulate IMPC cell clusters.\r\nMethods: HIF-1 activation was measured by EMSA and ELISA in MCF7 3-D spheroids and MCF7 monolayers.\r\nBinding of HIF-1a to MDR-1 gene promoter and modulation of P-glycoprotein (Pgp) expression was evaluated by\r\nChIP assay and FACS analysis, respectively. Intracellular doxorubicin retention was measured by spectrofluorimetric\r\nassay and drug cytotoxicity by annexin V-FITC measurement and caspase activity assay.\r\nResults: In MCF7 3-D spheroids HIF-1 was activated and recruited to participate to the transcriptional activity of\r\nMDR-1 gene, coding for Pgp. In addition, Pgp expression on the surface of cells obtained from 3-D spheroids was\r\nincreased. MCF7 3-D spheroids accumulate less doxorubicin and are less sensitive to its cytotoxic effects than\r\nMCF7 cells cultured as monolayer. Finally, HIF-1a inhibition either by incubating cells with 3-(5�-hydroxymethyl-2�-\r\nfuryl)-1-benzylindazole (a widely used HIF-1a inhibitor) or by transfecting cells with specific siRNA for HIF-1a\r\nsignificantly decreased the expression of Pgp on the surface of cells and increased the intracellular doxorubicin\r\naccumulation in MCF7 3-D spheroids.\r\nConclusions: MCF7 breast cancer cells cultured as 3-D spheroids are resistant to doxorubicin and this resistance is\r\nassociated with an increased Pgp expression in the plasma membrane via activation of HIF-1. The same\r\nmechanism may be suggested for IMPC drug resistance.
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