Combined Modulation of Tumor Metabolism by Metformin and Diclofenac in Glioma

Glioblastoma remains a fatal diagnosis. Previous research has shown that metformin, which is an inhibitor of complex I of the respiratory chain, may inhibit some brain tumor initiating cells (BTICs), albeit at dosages that are too high for clinical use. Here, we explored whether a combined treatment of metformin and diclofenac, which is a non-steroidal anti-inflammatory drug (NSAID) shown to inhibit glycolysis by interfering with lactate efflux, may lead to additive or even synergistic effects on BTICs (BTIC-8, -11, -13 and -18) and tumor cell lines (TCs, U87, and HTZ349). Therefore, we investigated the functional effects, including proliferation and migration, metabolic effects including oxygen consumption and extracellular lactate levels, and effects on the protein level, including signaling pathways. Functional investigation revealed synergistic anti-migratory and anti-proliferative effects of the combined treatment with metformin and diclofenac on BTICs and TCs. Signaling pathways did not sufficiently explain synergistic effects. However, we observed that metformin inhibited cellular oxygen consumption and increased extracellular lactate levels, indicating glycolytic rescue mechanisms. Combined treatment inhibited metformin-induced lactate increase. The combination of metformin and diclofenac may represent a promising new strategy in the treatment of glioblastoma. Combined treatment may reduce the effective doses of the single agents and prevent metabolic rescue mechanisms. Further studies are needed in order to determine possible side effects in humans

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo

Ibuprofen and Diclofenac Restrict Migration and Proliferation of Human Glioma Cells by Distinct Molecular Mechanisms

Background Non-steroidal anti-inflammatory drugs (NSAIDs) have been associated with anti-tumorigenic effects in different tumor entities. For glioma, research has generally focused on diclofenac; however data on other NSAIDs, such as ibuprofen, is limited. Therefore, we performed a comprehensive investigation of the cellular, molecular, and metabolic effects of ibuprofen and diclofenac on human glioblastoma cells. Methods Glioma cell lines were treated with ibuprofen or diclofenac to investigate functional effects on proliferation and cell motility. Cell cycle, extracellular lactate levels, lactate dehydrogenase-A (LDH-A) expression and activity, as well as inhibition of the Signal Transducer and Activator of Transcription 3 (STAT-3) signaling pathway, were determined. Specific effects of diclofenac and ibuprofen on STAT-3 were investigated by comparing their effects with those of the specific STAT-3 inhibitor STATTIC. Results Ibuprofen treatment led to a stronger inhibition of cell growth and migration than treatment with diclofenac. Proliferation was affected by cell cycle arrest at different checkpoints by both agents. In addition, diclofenac, but not ibuprofen, decreased lactate levels in all concentrations used. Both decreased STAT-3 phosphorylation; however, diclofenac led to decreased c-myc expression and subsequent reduction in LDH-A activity, whereas treatment with ibuprofen in higher doses induced c-myc expression and less LDH-A alteration. Conclusions This study indicates that both ibuprofen and diclofenac strongly inhibit glioma cells, but the subsequent metabolic responses of both agents are distinct. We postulate that ibuprofen may inhibit tumor cells also by COX- and lactate-independent mechanisms after long-term treatment in physiological dosages, whereas diclofenac mainly acts by inhibition of STAT-3 signaling and downstream modulation of glycolysis

Heterogeneity of Amino Acid Profiles of Proneural and Mesenchymal Brain-Tumor Initiating Cells

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo

Ibuprofen and diclofenac led to cell cycle arrest at different checkpoints.

<p>Human glioma cell lines HTZ-349 (A), A172 (B), and U87MG (C) were cultured in the presence of increasing concentrations of diclofenac (0.1, 0.2 mM) or ibuprofen (1, 2 mM) and respective DMSO controls. Cells were harvested for cell cycle analysis after 48 h of incubation. Proliferation was significantly reduced as measured by a reduced number of cells in S-phase in all conditions except 0.1 mM diclofenac in A172 as well as 0.1 and 0.2 mM diclofenac in HTZ-349. With diclofenac treatment, HTZ-349 and U87MG cells showed an accumulation of cells in the G2/M (U87MG) and S-phase (HTZ-349), whereas A172 arrested in the G1 phase. Ibuprofen generated accumulation of cells in the G1 phase in all cell lines. Additional treatment with 0.05–2 mM ibuprofen (D) confirmed dose-dependent accumulation of HTZ-349 in G1 accompanied by a decrease of cells in S- and G2/M-phase. Bar graphs show mean values of three independent experiments. Histograms are depicted in the supplements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s002" target="_blank">S2 Fig</a>) and show representative plots of each treatment condition.</p

IC₅₀ is distinct for diclofenac, ibuprofen, and ASA.

<p>Concentration-dependent cytotoxicity was investigated by treating the glioma cell lines HTZ-349, U87MG, and A172 in absence or presence of increasing concentrations of (A) ibuprofen (0.5–2.0 mM), (B) diclofenac (0.05–0.2 mM), or (C) ASA (0.05–0.2 mM) and DMSO as control in corresponding concentrations. The assays showed a significant linear concentration-dependent decrease of cell viability, which significantly differed between (A) ibuprofen (IC₅₀, 1 mM) and (B) diclofenac (IC₅₀, 0.1 mM). In contrast, (C) ASA had no significant effect on cell proliferation with given concentrations. Statistics: 95% CI, *** = 0.001 < p ≥ 0.0001, **** = p ≤ 0.0001.</p

Ibuprofen and diclofenac decrease migration.

<p>Spheroids of human glioma cell line HTZ-349 (5 x 10³ cells/well) were cultured in presence of increasing (A) ibuprofen (0.5–2 mM) or (B) diclofenac concentrations (0.05–0.2mM) and monitored for 30 h. During this time, pictures of cells migrating from the spheroids were taken periodically to monitor migratory capacity at treatment conditions (C) (scale bar = 500 μm). Migration was significantly inhibited by ibuprofen (A) in a time- and concentration-dependent manner. After 24 h, all concentrations demonstrated significantly greater migration over controls (95% CI, **** = p < 0.0001). (D) A high concentration of ibuprofen, 2 mM, resulted in significant migration inhibition noticeable by 6 h (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s003" target="_blank">S3A Fig</a>). Diclofenac resulted in similar (B), although less pronounced (D), effects. Measurement of attachment capacities after 0.1 mM ibuprofen (E) or 0.05 mM diclofenac (F) revealed adhesion deficits of HTZ-349 cells compared to controls within the first 30 minutes (95% CI, * = 0.05 > p ≤ 0.01, ** = 0.01 > p ≤ 0.001, *** = 0.001 > p ≤ 0.0001, **** = p < 0.0001.).</p

Ibuprofen and diclofenac decrease lactate levels and LDH activity.

<p>Human glioma cell lines HTZ-349, A172, and U87MG (10⁵ cells/well) were cultured for 24 h with increasing concentrations of ibuprofen or diclofenac as indicated, then supernatants were harvested for lactate level measurement (A–C). Measurements were normalized to cell number. Diclofenac treatment resulted in a significant decrease of lactate at all concentrations and in all cell lines. In contrast, ibuprofen cause a reduction of lactate accumulation only at concentrations of 2 mM in HTZ-349 and U87MG. These observations are reflected by LDH activity measurements (D) which exhibited decreased activity with increasing concentrations of diclofenac. LDH activity reduction with ibuprofen treatment occurred only at a concentration of 2 mM. Statistics: 95% CI, * = 0.05 > p ≤ 0.01, ** = 0.01 > p ≤ 0.001, *** = 0.001 > p ≤ 0.0001, **** = p < 0.0001.</p