A sphingosine kinase inhibitor induces cell death in temozolomide resistant glioblastoma cells

Abstract

Purpose: Sphingosine kinase is an oncogene that is up-regulated in several solid tumors. The product of the sphingosine kinase activity, sphingosine-1-phosphate is a potent mitogen involved in diverse cell processes such as cell survival and migration. Current standard therapy in the treatment of glioblastoma multiforme (GBM) is a combination of surgery, radiation, and chemotherapy using the drug temozolomide (TMZ). However, virtually all tumors become resistant to TMZ. Therefore, new drug targets are necessary. In this study, we investigated whether inhibiting sphingosine kinase could induce cell death in TMZ-resistant GBM cells.

Methods: To study TMZ resistance in vitro, we have generated TMZ-resistant cell lines from established GBM cells. We used a potent inhibitor of sphingosine kinase to study its effect on colony formation and cell growth in GBM cells with a limited dilution and WST assay. Moreover, cell death was determined by measuring caspase-3 activity using flow cytometry.

Results: A sphingosine kinase inhibitor reduced cell colony formation and activated caspase-3 in both TMZ-sensitive and resistant GBM cells.

Conclusion: Addition of a sphingosine kinase inhibitor to the standard chemotherapy regimen against GBM may be beneficial.

Fig. 1

Limiting dilution assay shows gain of resistance to TMZ in OTR cell lines of a D54MG cells and b U251 cells. Data are the mean ± SD of three different experiments and are expressed as survival fractions. c Real-time RT-PCR displays up-regulation of Sphk1 gene expression in the OTR lines of U251 and D54MG compared to the respective parental line. Data are the mean ± SD of duplicate determinations and are expressed as fold increase compared with parental cell line

Fig. 2

SKI reduces cell growth and colony formation and induces cell death. Treatment of D54MG (a) and U251 (b) cells with SKI inhibits colony formation in both the parental and OTR lines as measured with a limiting dilution assay. Data are the mean ± SD of three experiments performed on different days and are expressed as survival fractions. Effect of SKI on cell proliferation in U251 (c) and D54MG (d) cells were measured as described under “Methods”. Data are the mean ± SD of quadruplicate determinations and are displayed as absorption measured at OD 450 nm. Similar results were obtained in two additional experiments. e Caspase-3 activation in U251 cells upon SKI treatment was measured using flow cytometry. Similar results were obtained in two additional experiments