Abstract 5049: Cyclopamine modulates ABCG2 activity in glioblastoma side population cells

Abstract

Abstract The Sonic Hedgehog pathway (HH) has been implicated in the maintenance of stem cells in adult tissues. Abnormal HH activation is also associated with initiation of neoplasia by cancer stem cells. Cyclopamine has been used as an inhibitor of the HH, with potential effectiveness in several xenograft models, including gliomas. Side Population (SP) cells are stem cells with expression of ABCG2, a multidrug ABC transporter, able to extrude dyes as Hoechst 33342 (Ho342). SP cells, then, present low fluorescence after staining with Ho342. ABC transporters have been identified as drug-resistance proteins. HH regulates the expression of ABC transporters, and promotes multi-drug resistance by increasing drug efflux by the ABC transporters. Different research groups have shown that SP cells can be depleted following cyclopamine treatment, an observation that would support that SP cells are HH pathway-dependent. We hypothesized that cyclopamine might interact with ABCG2 and limit the number of SP cells. Therefore, we analyzed the effect of cyclopamine and temozolomide on the MXRA cell line (KB cells transfected with the full-length ABCG2 cDNA), and on five astrocytoma cell lines, both in the parental cell lines and in their SP cells. We first evaluated the modulatory effect of cyclopamine on ABCG2 function by measuring the intracellular accumulation of two substrates of ABCG2, Ho342 and mitoxantrone. Secondly, we analyzed the effect of these drugs in the phenotype of SP cells (the cells were incubated with only Ho342, or a combination of Ho342 and cyclopamine, or a combination of Ho342, cyclopamine and temozolomide; afterwards we analyzed the number of SP cells). We saw that cyclopamine significantly decreased the fraction of SP cells but not through a depletion of the SP compartment, but as a consequence of the molecular mechanisms involved in the affinity of cyclopamine and Ho342 for ABCG2, as cyclopamine modulated the ABCG2 transporter by increasing Ho342 and mitoxantrone uptake. Temozolomide had no effect on the SP cells. Therefore, cyclopamine, although decreases the SP fraction of cancer stem cells in glioblastoma cell lines, does not deplete the SP compartment, but modulates ABCG2 activity. 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 5049. doi:10.1158/1538-7445.AM2011-5049