The online version of this article (doi:10.1186/1476-4598-11-13) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
EL generated the breast cancer DoxoR cell lines and carried out the immunofluorescence, RIP-Chip, cell fractionation, western blotting, silencing and caspase activation experiments, and performed the cell toxicity and combination drug synergy experiments. TT carried out the RIP-Chip and GE microarray data analysis. GV participated together with EL in the polysomal HuR translocation experiment and discussed data. AMS performed the cell toxicity and combination drug synergy test. EL discussed the experiments and drafted the manuscript. AQ discussed the experiments and participated in critically revising the manuscript. AP conceived study, designed the experiments, supervised the work and wrote the manuscript. All authors read and approved the manuscript.
HuR, an RNA binding protein involved in the post-transcriptional regulation of a wide spectrum of mRNAs, has been demonstrated to be a determinant of carcinogenesis and tumor aggressiveness in several cancer types. In this study, we investigated the role of HuR in the apoptosis and in the chemoresistance induced by the widely used anticancer drug doxorubicin in human breast cancer cells (MCF-7).
We showed that HuR acts in the early phase of cell response to doxorubicin, being induced to translocate into the cytoplasm upon phosphorylation. Reducing HuR levels diminished the apoptotic response to doxorubicin. Doxorubicin-induced apoptosis was also correlated with the presence of HuR in the cytoplasm. Rottlerin, which was able to block HuR nuclear export, had correspondingly antagonistic effects with doxorubicin on cell toxicity. The proapoptotic activity of HuR was not due to cleavage to an active form, as was previously reported. In in vitro selected doxorubicin resistant MCF-7 cells (MCF-7/doxoR) overexpressing the multidrug resistance (MDR) related ABCG2 transporter, we observed a significant HuR downregulation that was paralleled by a corresponding downregulation of HuR targets and by loss of rottlerin toxicity. Restoration of HuR expression in these cells resensitized MCF-7/doxoR cells to doxorubicin, reactivating the apoptotic response.
The present study shows that HuR is necessary to elicit the apoptotic cell response to doxorubicin and that restoration of HuR expression in resistant cells resensitizes them to the action of this drug, thereby identifying HuR as a key protein in doxorubicin pharmacology.
Additional file 1: Figure S1. Doxorubicin induced apoptosis. Annexin-V FACS assay on MCF-7 cells treated with different doxorubicin (doxo) concentration for 18 h or not (untreated). The upper left dot plot indicates which area is occupied by necrotic, apoptotic or living cells respectively. Freeze and tow (F&T) sample was used as necrosis positive control and cycloexamide 10 μM (CXM) was used as apoptosis positive control. (PDF 143 KB)12943_2011_997_MOESM1_ESM.PDF
Additional file 2: Table S1. List of mRNAs bound to HuR in the presence of doxorubicin. The complete list of mRNAs precipitated with HuR during RIP-Chip. Probe indicates the Agilent reference number, gene symbol is the HGNC symbol, HuR fold enrichment is the fold enrichment of mRNA bound to HuR in comparison to cytoplasmic amount, AU-rich stability element is how many AREs are present in the 3'UTR of the mRNA according to TRANSTERM, description is gene annotation. (XLSX 65 KB)12943_2011_997_MOESM2_ESM.XLSX
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- Downregulation of HuR as a new mechanism of doxorubicin resistance in breast cancer cells
Antonino Maria Spartà
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