Phytochemical Regulation of Tumor Suppressive MicroRNA in Human Breast Cancer Cells
MicroRNA post-transcriptionally regulate more than half of the transcribed human genome and could be potential targets of anti-cancer therapeutics. The microRNA family, miR-34, is a component of the p53 tumor suppressor pathway and has been shown to mediate induction of cell cycle arrest, senescence, and apoptosis in cancer cells. Indole-3-carbinol (I3C) derived from cruciferous vegetables, artemisinin isolated from the sweet wormwood plant and artesunate derived from the carbonyl reduction of artemisinin effect components of the p53 pathway to growth arrest human cancer cells, implicating a potential role for miR-34 in their anti-proliferative effects. Flow cytometry and Taqman semi-quantitative PCR analysis of I3C, artemisinin and artesunate treated human breast cancer cells indicate all three phytochemicals upregulate miR-34a in a dose and time-dependant manner that correlates with a pronounced G1 cell cycle arrest. Western blot analysis revealed miR-34a upregulation correlates with induction of functional p53 by I3C as well as artesunate and artemisinin mediated decreases in estrogen-receptor alpha and the cyclin-dependant kinase CDK4, a known target of miR-34a inhibition. Luciferase assays in which cells were transfected with the miR-34a binding site of CDK4 mRNA attached to the firefly luciferase reporter gene confirmed miR-34a directly inhibits CDK4 expression in cells growth arrested by artemisinin or artesunate. Functional miR-34a appears critical for the anti-proliferative effects of I3C and artemisinin as transfection of non-translatable miR-34a inhibitors prevented I3C mediated growth inhibition and reversed artemisinin mediated down-regulation of CDK4. Artemisinin mediated down-regulation of ERα was also reversed in cells transfected with miR-34a inhibitors, implicating a novel role for miR-34a in the regulation of hormonal signaling. Transfection of dominant negative p53 prevented I3C upregulation of miR-34a in growth arrested cells containing wild-type p53 yet had no effect on artemisinin regulation of miR-34a, indicating a p53-indepedent mechanism of miR-34a regulation. Artemisinin and artesunate also upregulate miR-34a expression levels in breast cancer cell lines containing non-functional p53. All of these data suggest that miR-34a plays a critical role in the anti-proliferative effects of artemisinin, artesunate and indole-3-carbinol in human breast cancer cells. Such evidence elucidates the therapeutic potential of each phytochemical to ectopically express tumor suppressive microRNA while implicating the use of miR-34a expression levels to determine the efficacy of phytochemical treatment. Artemisinin, artesunate and I3C could represent an efficacious means of increasing tumor suppressive microRNA in vivo.