MicroRNA regulation of anthracycline drug resistance in leukemia through miR-221, miR-222, miR-26a, and miR-21

Drug resistance takes many forms including decreased drug influx, increased metabolism by cytochrome P450 enzymes, and increased drug efflux, with P-gp mediated efflux of chemotherapeutic drugs being one of the most common. Anthracyclines, which are prevalently used drugs in cancer treatment, each contain a sugar moiety which is important in the drug's efficacy. Through modification of the sugar moiety we propose to both overcome P-gp related drug resistance and determine if alternative forms of drug resistance exist. Through methods of chemical biology we created 25 derivatives of daunorubicin with varying alterations to the sugar structure. We showed that the activity of the DNR can be more or less effective through these changes. Molecular docking showed that changing the amino group to an azido group or conversion of a monosaccharide to a disaccharide made binding to P-gp less favorable. Further docking studies showed that alteration of the sugar group could change DNR's specificity from a topoisomerase2 poison to a topoisomerase1 poison. Additionally sugar modification will change the specificity of the compound during DNA interchelation resulting in changes to microRNA levels. The combination of these studies provides us with a better understanding of the critical elements of the sugar moiety of anthracyclines, and provides us with a starting point to design more effective anticancer drugs.; From the 25 derivatives created using chemical biology, we identified two new anthracycline analogs (ADNR, ADNR3) which avert P-gp binding and overcome P-gp-mediated drug resistance. To study other novel mechanisms for drug resistance in leukemia independent of P-gp, doxorubicin, daunorubicin, ADNR, and ADNR3 were used to induce a new type of drug resistance, which was associated with microRNA regulation.; A microRNA microarray assay demonstrated that 5 to 15 microRNAs were up-regulated by at least 1.5-fold, and 2 to 122 microRNAs were down-regulated by 2 to 4852-fold in drug induced resistant leukemia cells by doxorubicin (DOX), daunorubicin (DNR), ADNR, and ADNR3. miR-221 and miR-222 were consistently down-regulated by 2 to 15-fold in all four drug induced resistant cells. Low levels of miR-221 and miR-222 in these drug-resistant cells up-regulate 4 to 8-fold expression of Kit protein, a receptor tyrosine kinase for cell survival. Transfection of miR-221 and miR-222 in four drug-resistant cells regained drug sensitivity. This indicates that down-regulation of miR-221 and miR-222 and subsequent up-regulation of Kit provides a novel mechanism for drug resistance in leukemia. Application of microRNA may provide a novel tool to battle drug resistance in leukemia chemotherapy.; Through real time RT-PCR we also determined that miR-26a was down-regulated by 1.49 to 3.60-fold in doxorubicin and daunorubicin resistant K562 leukemia cells. These low levels of miR-26a in doxorubicin and daunorubicin resistant cells up-regulate the expression of cdk6 by 3.65 and 4.42-fold. The miR-26a/cdk6 regulation mechanism was confirmed through transfection of miR-26a into drug resistant cells where cdk6 expression levels were knocked out, and drug sensitivity was restored. The IC50 value for doxorubicin was decreased by 11.01-fold, and the IC50 value of daunorubicin was decreased by 12.97-fold. This indicates that down-regulation of miR-26a and subsequent up-regulation of cdk6 provides a novel mechanism for drug resistance. The regulation of cdk6 by miR-26a opens the door for further research into the regulation of cell cycle proteins by microRNA. This gives a better understanding into the role microRNA plays in drug resistance, and allows for application of microRNA as a tool to help battle drug resistance in leukemia.; Anthracycline treatment also up-regulates microRNA, including miR-21 which was up-regulated by 2.51 to 6.51-fold in the four drug resistant cell lines. miR-21 is of importance due to its regulation of the tumor suppressor, PTEN. This upre...