Alteration of glycosphingolipid levels and overexpression of GlcCer synthase are associated to acquired resistance to unrelated drugs in human glioma cells

Different studies support that glycosphingolipids (GSLs) act as important players in tumor biology, and emerging evidence suggests their involvement in drug resistance too. In tumor specimens from patients with scarce response to chemotherapy and in drug-resistant cancer cells, the activity of glucosylceramide synthase (GCS) and the levels of glucosylceramide (GlcCer) were found elevated. Moreover, inhibitors of GCS have been shown to increase the cellular levels of ceramide, a key player in the regulation of apoptosis, and reverse drug resistance. Notwithstanding, little is known on the involvement of GSLs in malignant gliomas. These are the most frequent and deadly human primary brain tumors, their intrinsic or acquired resistance limiting therapy effectiveness. In this study we investigated the possible involvement of GSLs in the resistance of T98G human glioblastoma cells to two unrelated drugs with clinical activity against gliomas, i.e. the microtubule-disrupting agent paclitaxel and the alkylating drug temozolomide. By selection with gradually increasing drug concentrations, we generated both a paclitaxel (Ptx-R) and a temozolomide (Tmz- R) resistant cell line. The analysis of resistance markers by immunoblotting showed that MDR1 (P-glycoprotein) was highly expressed in Ptx-R cells, but undetectable in T98G and Tmz-R ones. O6-methylguanine-DNA methyltransferase was expressed in all cells, with marked overexpression exclusively in Tmz-R cells. After labeling at equilibrium with 3H-sphingosine or 3H-serine, both Ptx-R and Tmz-R cells were characterized, with respect to sensitive T98G, by increased levels of GlcCer and gangliosides. In addition, the ceramide levels were found lower in resistant vs. sensitive cells. A semi-quantitative RT-PCR analysis showed that a higher expression of the mRNA for the GCS gene was evident in both Ptx-R and Tmz-R cells. The in vitro activity of GCS in both resistant cells was found significantly higher than in sensitive ones. Noteworthy, the levels of lactosyl-ceramide, the major neutral glycosphingolipid of T98G cells, were significantly lower in Tmz-R, but not in Ptx- R cells. This finding supports the role for MDR1 as the major GlcCer translocase required for neutral glycosphingolipid anabolism, as well as the existence of distinct metabolic pathways for ganglioside and neutral glycolipid biosynthesis. Altogether our data demonstrate that overexpression of GCS and alterations of glycosphingolipid level, with increased GlcCer and ganglioside content, are associated to T98G resistance to unrelated cytotoxic drugs. These variations occur independently of MDR1 expression, and are associated to the attenuation of ceramide levels. This suggests that the increase of specific glycosphingolipids might offer glioma cells a survival advantage,resulting in resistance to chemotherapeutic drugs.