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Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF)-beta2 and migration of glioma cells in vitro.

Seliger C, Leukel P, Moeckel S, Jachnik B, Lottaz C, Kreutz M, Brawanski A, Proescholdt M, Bogdahn U, Bosserhoff AK, Vollmann-Zwerenz A, Hau P - PLoS ONE (2013)

Bottom Line: Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide.Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate.Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Wilhelm Sander-NeuroOncology Unit, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT

Background: An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods: Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results: Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion: We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

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Glioma cell migration is mediated by THBS-1 and TGF-beta2.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).
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pone-0078935-g007: Glioma cell migration is mediated by THBS-1 and TGF-beta2.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).

Mentions: To investigate the functional relevance of THBS-1 and TGF-beta2 on glioma cell migration we performed in vitro migration assays after differential treatment of glioma cells. First, we examined the migratory capacity of siLDH-A-transfected HTZ-349 and U87 glioma cells in Boyden (Figure 7A) and Scratch Migration Assays (Figure 7B). Both cell lines showed significantly decreased migration after knockdown of LDH-A (and therefore decreased levels of lactate) in comparison to controls. Proliferation was not significantly changed after siLDH-A treatment during the time periods used for migration assays (Figure S1C, S1D). To verify the initiating capacity of THBS-1, we examined HTZ-349 and U87 glioma cell migration after knockdown of THBS-1 (Figure 7C). Again, both cell lines exhibited significantly reduced migration after siTHBS-1 treatment in Scratch Migration Assays. Proliferation was not markedly impaired after siTHBS-1 treatment (Figure S1E, S1F). Finally, we investigated how addition of synthetic THBS-1 and recombinant TGF-beta2 mediates glioma cell migration. Addition of 6 µg/ml THBS-1 completely rescued impaired migration after LDH-A knockdown (Figure 7D). Similar results were obtained in U87 (not shown). Accordingly, addition of 20 ng/ml TGF-beta2 also fully restored reduced glioma cell migration after LDH-A knockdown in HTZ-349 (Figure 7E) and U87 (Figure 7F) glioma cells, indicating a cascade starting from lactate that is relevant for the migratory capacity of glioma cells.


Lactate-modulated induction of THBS-1 activates transforming growth factor (TGF)-beta2 and migration of glioma cells in vitro.

Seliger C, Leukel P, Moeckel S, Jachnik B, Lottaz C, Kreutz M, Brawanski A, Proescholdt M, Bogdahn U, Bosserhoff AK, Vollmann-Zwerenz A, Hau P - PLoS ONE (2013)

Glioma cell migration is mediated by THBS-1 and TGF-beta2.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).
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pone-0078935-g007: Glioma cell migration is mediated by THBS-1 and TGF-beta2.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).
Mentions: To investigate the functional relevance of THBS-1 and TGF-beta2 on glioma cell migration we performed in vitro migration assays after differential treatment of glioma cells. First, we examined the migratory capacity of siLDH-A-transfected HTZ-349 and U87 glioma cells in Boyden (Figure 7A) and Scratch Migration Assays (Figure 7B). Both cell lines showed significantly decreased migration after knockdown of LDH-A (and therefore decreased levels of lactate) in comparison to controls. Proliferation was not significantly changed after siLDH-A treatment during the time periods used for migration assays (Figure S1C, S1D). To verify the initiating capacity of THBS-1, we examined HTZ-349 and U87 glioma cell migration after knockdown of THBS-1 (Figure 7C). Again, both cell lines exhibited significantly reduced migration after siTHBS-1 treatment in Scratch Migration Assays. Proliferation was not markedly impaired after siTHBS-1 treatment (Figure S1E, S1F). Finally, we investigated how addition of synthetic THBS-1 and recombinant TGF-beta2 mediates glioma cell migration. Addition of 6 µg/ml THBS-1 completely rescued impaired migration after LDH-A knockdown (Figure 7D). Similar results were obtained in U87 (not shown). Accordingly, addition of 20 ng/ml TGF-beta2 also fully restored reduced glioma cell migration after LDH-A knockdown in HTZ-349 (Figure 7E) and U87 (Figure 7F) glioma cells, indicating a cascade starting from lactate that is relevant for the migratory capacity of glioma cells.

Bottom Line: Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide.Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate.Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology and Wilhelm Sander-NeuroOncology Unit, University Hospital Regensburg, Regensburg, Germany.

ABSTRACT

Background: An important phenomenon observed in glioma metabolism is increased aerobic glycolysis in tumor cells, which is generally referred to as the Warburg effect. Transforming growth factor (TGF)-beta2, which we previously showed to be induced by lactic acid, is a key pathophysiological factor in glioblastoma, leading to increased invasion and severe local immunosuppression after proteolytic cleavage from its latency associated peptide. In this study we tested the hypothesis, that lactate regulates TGF-beta2 expression and glioma cell migration via induction of Thrombospondin-1 (THBS-1), a TGF-beta activating protein.

Methods: Lactate levels were reduced by knockdown of LDH-A using specific small interfering RNA (siRNA) and competitive inhibition of LDH-A by sodium oxamate. Knockdown of THBS-1 was performed using specific siRNA. Western Blot, qRT-PCR, and ELISA were used to investigate expression levels of LDH-A, LDH-B, TGF-beta2 and THBS-1. Migration of cells was examined by Spheroid, Scratch and Boyden Chamber assays.

Results: Knockdown of LDH-A with subsequent decrease of lactate concentration leads to reduced levels of THBS-1 and TGF-beta2 in glioma cells. Lactate addition increases THBS-1 protein, leading to increased activation of TGF-beta2. Inhibition of THBS-1 reduces TGF-beta2 protein and migration of glioma cells. Addition of synthetic THBS-1 can rescue reduced TGF-beta2 protein levels and glioma cell migration in siLDH-A treated cells.

Conclusion: We define a regulatory cascade between lactate, THBS-1 and TGF-beta2, leading to enhanced migration of glioma cells. Our results demonstrate a specific interaction between tumor metabolism and migration and provide a better understanding of the mechanisms underlying glioma cell invasion.

Show MeSH
Related in: MedlinePlus