Abstract 1721: Resistance to silvestrol is mediated by MDR1/Pgp over-expression in a lymphoblastic leukemia cell line and is reversible by treatment with rapamycin

Abstract

Abstract We previously showed that silvestrol, a translation initiation inhibitor, has potent cytotoxic activity in chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) B-cells in vitro and in vivo (Blood. 2009; 113(19):4656-66). To study potential mechanisms of silvestrol resistance, we generated a silvestrol-resistant B-leukemia cell line as a model system for B-cell leukemias. The 697 ALL cell line was incubated with step-wise increasing concentrations of silvestrol to generate a cell line (697-R) resistant to 80 nM silvestrol. In contrast, the LC50 (concentration lethal to 50%) of silvestrol in the parental 697 cell line is approximately 5 nM. 697-R cells retained genotypic and immunophenotypic features of the parental 697 cell line. One common mechanism of drug resistance in cancer is the over-expression of the multidrug resistance gene ABCB1 and its protein product, multidrug resistance-1/permeability glycoprotein (MDR1/Pgp). Using RT-PCR and flow cytometry, we confirmed increased MDR1 mRNA and protein expression in 697-R cells compared to parental 697 cells. Consistent with MDR1 upregulation, 697-R cells also showed resistance to the MDR1 substrates vincristine and depsipeptide, but not to the non-MDR1 substrate flavopiridol. We next assessed the functional activity of MDR1 in 697-R by measuring efflux of the fluorescent MDR1 substrate rhodamine 123 by flow cytometry. Rhodamine 123 was nearly completely eliminated from 697-R cells within 2 hours, but was fully retained in parental 697 cells. The common MDR1 inhibitors verapamil and cyclosporine inhibited rhodamine efflux from 697-R cells. As this type of multidrug resistance can be mediated by several genes, we used MDR1-specific siRNA to determine whether over-expression of MDR1 is responsible for rhodamine efflux. At 48 hours post-transfection, MDR1 siRNA-transfected cells showed significantly reduced efflux of rhodamine 123 compared to scrambled and irrelevant controls. It has previously been reported that the mTOR inhibitor rapamycin can also block MDR1 function. We therefore tested this using the 697-R cell line. Similar to verapamil, rapamycin was also able to inhibit the efflux of rhodamine 123 from these cells. Furthermore, rapamycin was also able to restore silvestrol sensitivity to 697-R cells. In conclusion, we show that MDR1/Pgp over-expression is at least one mechanism of silvestrol resistance in cell lines, and known MDR1 inhibitors including rapamycin can reverse this resistance in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1721. doi:10.1158/1538-7445.AM2011-1721