21 research outputs found
Ibuprofen and diclofenac decrease lactate levels and LDH activity.
<p>Human glioma cell lines HTZ-349, A172, and U87MG (10<sup>5</sup> 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
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
IC<sub>50</sub> 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 (IC<sub>50</sub>, 1 mM) and (B) diclofenac (IC<sub>50</sub>, 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 10<sup>3</sup> 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 proliferation.
<p>Proliferation was analyzed using crystal violet staining and results were verified via the CyQuant Direct Cell Proliferation assay in HTZ-349. (A) Starting at 72 h, all ibuprofen concentrations (0.5, 1, 2 mM) resulted in significant reduction of HTZ-349 proliferation (compared to non-treated Ctrl, 95% CI, p < 0.05). 2 mM ibuprofen caused significant proliferation inhibition at 24 h (0.01 > p ≤ 0.001) and 48 h (p < 0.0001). (B) Proliferation decrease was verified in CyQuant measurements. Additionally, physiological ibuprofen concentrations (0.05–0.3 mM) were analyzed. Most demonstrated significant proliferation decrease after long-term treatment (144 h) compared to the non-treated controls (0.3–2 mM = p < 0.0001, 0.1 mM = 0.01 > p ≤ 0.001; 95% CI). (C) Similar proliferation reducing effects were obtained with diclofenac at respective concentrations (0.05, 0.1, 0.2 mM) (compared to DMSO control, 95% CI, p < 0.05). The highest concentration of 0.2 mM reached significant proliferation inhibition at 24 and 48 h compared to control (p < 0.0001). (D) CyQuant assay measurements of HTZ-349 treated with diclofenac were broadly in compliance with previously obtained results by crystal violet staining. (E) In contrast, ASA exhibited neither concentration- nor time-dependent effects, and only unspecific effects were observed at 96 h (0.05 mM: 0.05 > p ≤ 0.01) and 120 h (0.2 mM: p < 0.0001). (F) Corresponding CyQuant assay measurements of HTZ-349 treated with ASA verified the results. Treatment of A172 and U87MG cells showed similar results (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s011" target="_blank">S1 Fig</a>).</p
Ibuprofen and diclofenac affect STAT-3 signalling differentially.
<p>Transcription factors within the STAT-3 signalling pathway were investigated by Western blot (40 μg of protein) after incubation with increasing ibuprofen (0.5, 1, 2 mM) or diclofenac concentrations (0.05, 0.1, 0.2 mM) for 24 h. (A) Ibuprofen and diclofenac reduced STAT-3 phosphorylation in HTZ-349 cells without affecting total STAT-3 levels (for detailed quantification see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s005" target="_blank">S5 Fig</a>). The STAT-3 downstream target c-myc was significantly upregulated by ibuprofen (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s005" target="_blank">S5A Fig</a>), whereas diclofenac blocked pSTAT-3 and c-myc in a concentration-dependent manner (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s005" target="_blank">S5B Fig</a>). LDH-A, a direct target of c-myc, showed negative trends, but not to significant extent. Corresponding quantitative RT-PCR revealed significant LDH-A transcript decrease with diclofenac (0.2 mM, compared to control and DMSO control, 95% CI, * = 0.05 > p ≤ 0.01). Ibuprofen (2 mM) had no impact on LDH-A expression (B). Similar observations were made with ASA (0.2 mM). Corresponding results for A172 and U87MG were generated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s006" target="_blank">S6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140613#pone.0140613.s007" target="_blank">S7</a> Figs).</p
Glioma cell migration is enhanced after treatment with sodium lactate.
<p>HTZ-349 (A) and U87 (B) glioma cells were treated with 20 mM sodium lactate (pH 7.4) and HCl (pH 7.1) was used as control. 24 hours after treatment cell migration was analyzed using Boyden Chamber assays. The Y-axis indicates number of migrated cells. Treatment with lactate leads to a highly significant increase in glioma cell migration (U87 and HTZ-349, p < 0.001***). </p
Glioma cell lines express LDH-A and secrete lactate.
<p>Glioma cell lines HTZ-349 and U87 were cultured under comparable conditions. Using qRT-PCR, mRNA levels of LDH-A were determined using specific primers for LDH-A (A). Additionally, total protein was isolated and LDH-A and LDH-B protein expression was determined in Western Blot analysis (B). Glioma cell lines have increased mRNA levels of LDH-A in comparison to fibroblasts and human brain RNA (A, p < 0.05*) and show increased protein expression, as do a melanoma cell line (MelIm) and a prostatic cancer cell line (PC3) (B). To examine lactate expression cell culture supernatants (2*10<sup>5</sup> cells/well) were collected and lactate levels were measured (C). Glioma cell lines secrete higher levels of lactate if compared to fibroblasts (U87, p < 0.01**; HTZ-349 p < 0.05*). </p
Lactate regulates TGF-beta2 protein.
<p>Specific siRNA against LDH-A was used to reduce extracellular lactate levels and to investigate consecutive changes in TGF-beta2 expression. siLDH-A reduces LDH-A (p < 0.01**) and TGF-beta2 (p< 0.05*) mRNA expression significantly 72 hours after treatment (A). In TGF-beta2 Western Blot (B) and ELISAs (C, D) siLDH-A reduces TGF-beta2 protein expression with a maximum reduction 72 hours after treatment (p< 0.001***). Results were normalized to control. Treatment with 20 mM lactic acid (pH 7.1) as well as 20 mM sodium lactate (pH 7.4) increases TGF-beta2 mRNA (E). Expression of TGF-beta2 protein 24 hours after treatment is significantly increased by lactic acid, lactate and HCl (F, lactic acid p < 0.05*; sodium lactate p < 0.01**, HCl p < 0.05*). </p
Glioma cell migration is reduced after competitive inhibition of LDH-A.
<p>Sodium oxamate was used for competitive inhibition of LDH. LDH activity was determined using the cytotox assay (Promega, Germany). Treatment with increasing doses of sodium oxamate (5 mM-75 mM) significantly reduces LDH activity 24 hours after treatment (HTZ, 349 p < 0.01** for 25mM, 50 mM and 75 mM sodium oxamate; U87, p < 0.001*** for 25mM, 50 mM and 75 mM sodium oxamate, fibroblasts, p < 0.01** for 10 mM and p < 0.001*** for 25mM, 50 mM and 75 mM sodium oxamate). HTZ 349 and U87 showed significantly higher LDH activity compared to fibroblasts (HTZ-349 p < 0.01<sup>##</sup>, U87 p < 0.001<sup>###</sup>) (A). Lactate levels were measured in cell culture supernatants 24 hours after treatment with increasing doses of sodium oxamate (10 mM-75 mM). Treatment with sodium oxamate leads to a dose- (B) and time-dependent reduction (C) of extracellular lactate levels. Treatment with 25 mM and 50 mM sodium oxamate significantly reduces HTZ-349 (D) and U87 (E) glioma cell migration starting 24 hours after treatment (HTZ, 349 p < 0.001*** at 24 h and 32 h for 25 and 50 mM sodium oxamate; U87, p < 0.05* at 24 h, p< 0.01** at 32 h for 50 mM sodium oxamate). (F) Boyden chamber assay showing similar effects as in (D) and (E) (U87, p< 0.001***; HTZ-349, p < 0.001***). The Y-axis indicates the number of migrated cells. Results were normalized to control.</p