Targeting cellular metabolism using rapamycin and/or doxycycline enhances anti-tumour effects in human glioma cells

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.

Inhibition of Metabolic Shift can Decrease Therapy Resistance in Human High-Grade Glioma Cells

“…It was clearly demonstrated in different studies that cancer bioenergetics is changed with dynamic alterations at metabolic level [13]. In our previous work, we tested TMZ with combined metabolic inhibitors, however, glutaminase inhibitors have not been studied [22]. It is well-known that beside glucose other important substrates can fuel bioenergetic mechanisms in mammalian cells.…”

“…All Materials were Purchased from Merck-Sigma-Aldrich, except where it is Indicated in the Text Human glioma cell lines -U373 Uppsala (U373-U; ECACC-08061901), U251 and U87 (ECACC-09063001, with PTEN, NF-1, p53 and MSH2 mutations; and ATCC-HTB-14, characteristic mutations in PTEN, NF-1 and Notch-2, respectively) were maintained in DMEM high glucose medium (Biosera) supplemented with 10% foetal bovine serum (FBS; Biosera), 2 mM L-glutamine (Biosera) and 100 UI/ml penicillin-streptomycin (Biosera) at 37°C with 5% CO 2 . After seeding, the cells were treated with different drugs (rapamycin-RAPA, 50 ng/ml; doxycycline-DOXY, 10 μM; temozolomide-TMZ, 100 μM; etomoxir-ETO, 50 μM; chloroquine-CHL, 50 μM and bis-2-(5-phenylacetoamido-1,3,4thiadiazol-2-yl)-ethyl sulfide-BPTES, 10 μM) and their combinations in 96-well plates (2-5 × 10 3 cells/well for proliferation tests) or in T25 flasks (3-6 × 10 5 cells/flaskfor Western blot experiments) for 72 h. The concentration of the drugs was applied based on our previous publications [22]. Lower dose than IC50 level was applied in drug combinations, these concentrations were defined in accordance with previously published data on IC50 [23][24][25][26].…”

“…Percentage of the cell proliferation was given relative to control samples. To analyse the additive or synergistic effects of different drug combinations the Combination Index (CI) was calculated as we described previously [22].…”

<p>Combined Delivery of Temozolomide and siPLK1 Using Targeted Nanoparticles to Enhance Temozolomide Sensitivity in Glioma</p>

“…[39][40][41][42] Although treatment using surgery, postoperative chemotherapy, and radiotherapy have advanced significantly, patients' prognosis is still poor, with an average survival rate of approximately one year after diagnosis. [43][44][45][46][47] TMZ is the first line chemotherapy for glioma. Although it is used widely to treat patients with glioma, its effectiveness in treatment is not very high because of TMZ resistant glioma.…”

<p>Emerging Roles and Therapeutic Interventions of Aerobic Glycolysis in Glioma</p>

“…122 In addition, the expression of glycolytic enzymes, including HKII and PKFP, were modulated by mTOR signaling as consequences of metabolic adaptation. 123 As one of the two main forms of mTOR, it has been verified that mTORC1 was paralleled with the process of glycolysis. 124 Besides, mTORC2 targeted the c-myc/GFAT1 axis to regulate the interaction between glycolysis and glutaminolysis in glioma.…”