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*10⁵ 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 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 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^##, U87 p < 0.001^###) (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

Glioma cell migration is mediated by THBS-1 and TGF-beta2.

<p>Boyden Chamber assays of HTZ-349 and U87 glioma cells 24 hours after treatment with 0.1 µM siLDH-A show a significant inhibition of migration (A, U87 p < 0.001***; HTZ-349 p < 0.01**). The Y-axis indicates the number of migrated cells. Scratch Migration assays verified these results (B-F). Here, the Y-axis indicates the area of an artificial gap in the confluent cell monolayer. Inhibition of LDH-A by siRNA yields similar results (B) as in the Boyden chamber assay. THBS-1 knockdown also diminishes HTZ-349 and U87 migration (C, HTZ-349 p < 0.01**; U87 p < 0.001***). Addition of 6 µg/ml recombinant THBS-1 (D) and 20 ng/ml TGF-beta2 (E, F) can fully rescue impaired migration after LDH-A knockdown (HTZ-349, p < 0.001*** for decrease of migration after siLDH-A, p < 0.05*^,# for induction of migration after treatment with TGF-beta2; U87, p < 0.01** for decrease of migration after siLDH-A, p < 0.05* for induction of migration after treatment with TGF-beta2 in mock- and p < 0.001^### for induction of migration in siLDHA-transfected TGF-beta2 treated cells). </p

Knockdown of THBS-1 down-regulates TGF-beta2 at the protein level.

<p>0.1 µM of siTHBS-1 significantly inhibit THBS-1 at the mRNA level as assessed in qRT-PCR (A, p< 0.05*). Western Blot analysis showed markedly reduced THBS-1 protein levels over 96 hours after treatment with siTHBS-1, with a maximum reduction 72 hours after treatment (B). In TGF-beta2 ELISAs siTHBS-1 causes significant down-regulation of TGF-beta2 protein in HTZ-349 glioma cells with a maximum reduction at 72 hours (C, p < 0.05*). Decreased levels of TGF-beta2 protein after LDH-A knockdown (D, mock/siLDH-A p < 0.05*) can be rescued by addition of increasing doses of synthetic THBS-1 protein (siLDH-A/siLDH-A+6 µg/ml THBS-1, p < 0.05^#). Lactic acid and sodium lactate fail to significantly induce TGF-beta2 expression after transfection with siTHBS-1 (E, significance is indicated as compared to mock treated cells with standard medium, p < 0.05*). </p

Regulation of THBS-1 expression by lactate.

<p>mRNA levels of THBS-1 were examined in qRT-PCR using specific primers for THBS-1. Treatment of glioma cells with 0.1 µM siLDH-A significantly suppresses THBS-1 at the mRNA level in HTZ-349 and U87 glioma cells (A, HTZ-349 p < 0.001***; U87 p < 0.05*). Western blot analysis was used to determine LDH-A and THBS-1 protein expression after treatment with siLDH-A. β-Actin was used as a loading control. Suppression of LDH-A also leads to a reduction of THBS-1 protein in HTZ-349 (B) and U87 (C) glioma cells. Further, TGF-beta2 expression and pSmad2 are markedly suppressed after siLDH-A treatment in HTZ-349 glioma cells (B). Treatment with 20 mM sodium lactate (pH 7.4) or 20 mM lactic acid (pH 7.1) significantly induces THBS-1 in HTZ-349 (D) and U87 (E) glioma cells at the mRNA level 24 hours after treatment (HTZ-349 p < 0.05^*; U87 for lactic acid p < 0.01**, for sodium lactate p < 0.001***). Treatment of siLDH-A transfected HTZ-349 glioma cells with sodium lactate and lactic acid can fully rescue impaired THBS-1 expression (HTZ-349 p < 0.05^# for sodium lactate and lactic acid after siLDH-A transfection) (D). Western Blot analysis confirmed induction of THBS-1 protein in U87 glioma cells after treatment with lactic acid and sodium lactate (F).</p