Temozolomide down-regulates P-glycoprotein expression in glioblastoma stem cells by interfering with the Wnt3a/GSK3/β-catenin pathway

Background. Glioblastoma multiforme stem cells display a highly chemoresistant phenotype, whose molecular basis were poorly known. We aim to clarify this issue and to investigate the effects of temozolomide on chemoresistant stem cells. Methods. A panel of human glioblastoma cultures, grown as stem cells (neurospheres) and adherent cells, were used. Results. Neurospheres had a multidrug resistant phenotype compared with adherent cells. Such chemoresistance was overcome by apparently non-cytotoxic doses of temozolomide, which chemosensitizes glioblastoma cells to doxorubicin, vinblastine and etoposide. This effect was selective for P-glycoprotein (Pgp) substrates and for stem cells, leading to investigate whether there was a correlation between the expression of Pgp and the activity of typical stemness pathways. We found that Wnt3a and ABCB1, which encodes for Pgp, were both highly expressed in glioblastoma stem cells and reduced by temozolomide. By methylating the CpG islands in the Wnt3a gene promoter, temozolomide decreased the expression of Wnt3a in glioblastoma stem cells, disrupted the glycogen synthase-3 kinase/β-catenin axis, prevented the transcriptional activation of ABCB1 and lowered the amount of Pgp. Wnt3a overexpression was sufficient to transform adherent cells into neurospheres and to simultaneously increase proliferation and ABCB1 expression. On the contrary glioblastoma stem cells silenced for Wnt3a lost the ability to form neurospheres and reduced at the same time the proliferation rate and the ABCB1 levels. Conclusions. Our work suggests that Wnt3a is an autocrine mediator of stemness, proliferation and chemoresistance in human glioblastoma and that temozolomide may chemosensitize the stem cells population by down-regulating the Wnt3a signalling.