Fingolimod Inhibits C6 Rat Glioma Proliferation and Migration, Induces Sub-G1 Cell Cycle Arrest, Mitochondrial and Extrinsic Apoptosis In Vitro and Reduces Tumour Growth In Vivo

Abstract

Glioblastoma multiforme (GBM), the most prevalent brain tumour, is universally fatal. GBM cells exhibit cell cycle disruption and treatment resistance, remarking an urgent need for newer treatments. Fingolimod, a sphingosine-1-phosphate receptor modulator, has been reported to have anti-cancer effects. This study investigated the therapeutic potentials of fingolimod in rat C6 cells and pursued the involved mechanism(s). Cell survival, proliferation, migration, and morphology of fingolimod-treated C6 cells were evaluated using MTT, soft-agar colony formation, wound-healing, and Giemsa staining assays. Apoptosis was investigated through acridine orange/ethidium bromide (AO/EB) and annexin V staining, and flow cytometry analysed the cell cycle. Quantitative reverse transcription PCR and western blotting were used to evaluate gene and protein expressions. An intracranial C6 rat model validated the anti-tumour effect of fingolimod. Fingolimod significantly reduced the survival and colonies of the C6 cells and delayed their gap closure. Cell shrinkage coupled with AO/EB and PI staining of the fingolimod-treated cells indicated apoptosis, subsequently confirmed by measuring the expression levels of the candidate genes involved in apoptosis and cell cycle, such as Bax/Bcl2, P53, Cytochrome C and Caspases 9/3, Fas, Fadd, Tnfrsf1a, Cdkn1a, and Ccnd1, at RNA and protein levels, indicating both extrinsic and mitochondrial apoptosis and cell cycle arrest at sub-G1 phase in fingolimod-treated cells. Furthermore, treating rats bearing intracranial C6 tumours with fingolimod led to significant suppression of intracranial tumour growth. Based on our findings, cell cycle arrest and apoptosis contribute to fingolimod antitumor effects.