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Fingolimod Inhibits C6 Rat Glioma Proliferation and Migration, Induces Sub-G1 Cell Cycle Arrest, Mitochondrial and
Safura Pournajaf1, Nastaran Afsordeh1, Hadi Bayat2
1Department of Physiology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
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.
Insights
Fingolimod effectively combats glioblastoma multiforme (GBM) by inducing apoptosis and cell cycle arrest in cancer cells. This study demonstrates fingolimod
Area of Science:
- Neuro-oncology
- Cancer Biology
- Pharmacology
Background:
- Glioblastoma multiforme (GBM) is an aggressive brain tumor with poor prognosis.
- GBM exhibits cell cycle dysregulation and resistance to current therapies, necessitating novel treatment strategies.
- Fingolimod, a sphingosine-1-phosphate receptor modulator, shows potential anti-cancer properties.
Purpose of the Study:
- To investigate the therapeutic efficacy of fingolimod against rat C6 glioblastoma cells.
- To elucidate the underlying mechanisms of fingolimod's anti-cancer effects, including apoptosis and cell cycle modulation.
- To validate fingolimod's anti-tumor activity in an in vivo intracranial glioblastoma model.
Main Methods:
- Cell viability, proliferation, and migration assays (MTT, soft-agar, wound-healing).
- Apoptosis assessment via AO/EB, Annexin V staining, and flow cytometry.
- Cell cycle analysis using flow cytometry.
- Gene and protein expression analysis (RT-PCR, Western blotting) of apoptosis- and cell cycle-related markers.
- In vivo efficacy study in an intracranial C6 rat tumor model.
Main Results:
- Fingolimod significantly inhibited C6 cell survival, proliferation, and migration.
- Apoptosis was induced in fingolimod-treated cells, evidenced by morphological changes and molecular markers (Bax/Bcl2, Caspases, Cytochrome C).
- Fingolimod caused cell cycle arrest at the sub-G1 phase.
- Fingolimod treatment suppressed tumor growth in rats with intracranial C6 gliomas.
Conclusions:
- Fingolimod exhibits significant anti-tumor effects against glioblastoma.
- Apoptosis and cell cycle arrest are key mechanisms mediating fingolimod's efficacy.
- Fingolimod represents a promising therapeutic agent for glioblastoma treatment.
