Abstract
BackgroundGlioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, thus gliomas are mainly incurable diseases. Tumour cells can adapt rapidly to alterations during therapeutic treatments related to their metabolic rewiring and profound heterogeneity in tissue environment. Renewed interests aim to develop effective treatments targeting angiogenesis, kinase activity and/or cellular metabolism. mTOR (mammalian target of rapamycin), whose hyper-activation is characteristic for many tumours, promotes metabolic alterations, macromolecule biosynthesis, cellular growth and survival. Unfortunately, mTOR inhibitors with their lower toxicity have not resulted in appreciable survival benefit. Analysing mTOR inhibitor sensitivity, other metabolism targeting treatments and their combinations could help to find potential agents and biomarkers for therapeutic development in glioma patients.MethodsIn vitro proliferation assays, protein expression and metabolite concentration analyses were used to study the effects of mTOR inhibitors, other metabolic treatments and their combinations in glioma cell lines. Furthermore, mTOR activity and cellular metabolism related protein expression patterns were also investigated by immunohistochemistry in human biopsies. Temozolomide and/or rapamycin treatments altered the expressions of enzymes related to lipid synthesis, glycolysis and mitochondrial functions as consequences of metabolic adaptation; therefore, other anti-metabolic drugs (chloroquine, etomoxir, doxycycline) were combined in vitro.ResultsOur results suggest that co-targeting metabolic pathways had tumour cell dependent additive/synergistic effects related to mTOR and metabolic protein expression patterns cell line dependently. Drug combinations, especially rapamycin + doxycycline may have promising anti-tumour effect in gliomas. Additionally, our immunohistochemistry results suggest that metabolic and mTOR activity alterations are not related to the recent glioma classification, and these protein expression profiles show individual differences in patients’ materials.ConclusionsBased on these, combinations of different new/old drugs targeting cellular metabolism could be promising to inhibit high adaptation capacity of tumour cells depending on their metabolic shifts. Relating to this, such a development of current therapy needs to find special biomarkers to characterise metabolic heterogeneity of gliomas.
Highlights
Glioma is the most common highly aggressive, primary adult brain tumour
Cell cultures and different reagents U251 (ECACC-09063001, characteristic glioma cell specific mutations in phosphatase and tensin homolog (PTEN), neurofibromatosis 1 (NF-1), p53, MutS protein homolog 2 (MSH2)), U87 (ATCCHTB-14, characteristic glioma cell specific mutations in PTEN, NF-1 and neurogenic locus notch homolog protein 2 (Notch-2)), U373 Uppsala (U373-U; ECACC-08061901) human glioma cells were cultured and treated in DMEM high glucose medium supplemented with 10% foetal bovine serum (FBS; HyClone), 2 mM L-glutamine and 100 UI/mL penicillin–streptomycin at 37 °C in a 5% CO2 atmosphere
Using different assays cells were plated onto 96-well plates (2–5 × 103 cells/well for proliferation tests) or into T25 flasks (3–6 × 105 cells/flask—for liquid chromatography-mass spectrometry (LC–MS) and Western blot experiments) and treated with different drugs and their combinations for 72 h, in combinations different drugs were added at the same time to the cell cultures. 1% dimethyl sulfoxide (DMSO) was added to control cells and was used as a vehicle for some of the compounds as it was required
Summary
Glioma is the most common highly aggressive, primary adult brain tumour. Clinical data show that therapeutic approaches cannot reach the expectations in patients, gliomas are mainly incurable diseases. Based on the World Health Organization (WHO) Classification of Central Nervous System Tumours, both low grade and high grade gliomas can be divided into two categories according to the presence or absence of IDH1/2 mutations. Other lower grade gliomas have better prognosis (median survival rate is approximately 5 years), recently gliomas have been considered as mainly incurable diseases. These led to continuous interest to develop more effective treatments targeting for example malignancy related angiogenesis [4], kinase activity and cellular metabolism [5, 6]
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