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Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus

Glycolysis and lactate production are augmented under hypoxic conditions.(a) Relative change in intracellular glucose concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, before and during inhibition of glucose uptake with 20 μM Cytochalasin B. (b) Rate of fall in intracellular glucose concentration, after inhibition of glucose uptake with Cytochalasin B in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a significant increase in glycolytic activity, as indicated by the increased rate of fall in glucose. (c) Relative change in intracellular lactate concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, during inhibition of lactate efflux via MCT1 with 300 nm AR-C155858. (d) Rate of change in intracellular lactate concentration, after inhibition of lactate transport in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a robust increase in the rate of lactate production. (e) Relative change in the RNA level of GLUT1 and LDH1 in MCF-7 cells after three days under hypoxic conditions. Data are represented as mean ± SEM.
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f5: Glycolysis and lactate production are augmented under hypoxic conditions.(a) Relative change in intracellular glucose concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, before and during inhibition of glucose uptake with 20 μM Cytochalasin B. (b) Rate of fall in intracellular glucose concentration, after inhibition of glucose uptake with Cytochalasin B in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a significant increase in glycolytic activity, as indicated by the increased rate of fall in glucose. (c) Relative change in intracellular lactate concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, during inhibition of lactate efflux via MCT1 with 300 nm AR-C155858. (d) Rate of change in intracellular lactate concentration, after inhibition of lactate transport in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a robust increase in the rate of lactate production. (e) Relative change in the RNA level of GLUT1 and LDH1 in MCF-7 cells after three days under hypoxic conditions. Data are represented as mean ± SEM.

Mentions: To investigate the significance of CAIX-mediated facilitation of lactate/H+ transport capacity for cancer cell metabolism and propagation, we determined changes in glycolysis and lactate production during hypoxia on the single cell level. We measured changes in intracellular glucose and lactate concentration in individual MCF-7 breast cancer cells, incubated three days under normoxic or hypoxic conditions, with FLIP12glu-700 μΔ6 and Laconic, respectively. Under hypoxic conditions, the glycolytic rate increased to 170%, as measured by the rate of decrease in intracellular glucose during inhibition of glucose import by 20 μM Cytochalasin B (Fig. 5a,b; ref. 34.)Lactate production increased to 162% under these conditions, as measured by the rate of increase in intracellular lactate during inhibition of lactate efflux with 300 nm AR-C155858 (Fig. 5c,d; ref. 35). Furthermore the mRNA levels of the glucose transporter GLUT1 and lactate dehydrogenase LDH-1, two markers for glycolytic metabolism, increased under hypoxic conditions (Fig. 5e).


Hypoxia-induced carbonic anhydrase IX facilitates lactate flux in human breast cancer cells by non-catalytic function.

Jamali S, Klier M, Ames S, Barros LF, McKenna R, Deitmer JW, Becker HM - Sci Rep (2015)

Glycolysis and lactate production are augmented under hypoxic conditions.(a) Relative change in intracellular glucose concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, before and during inhibition of glucose uptake with 20 μM Cytochalasin B. (b) Rate of fall in intracellular glucose concentration, after inhibition of glucose uptake with Cytochalasin B in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a significant increase in glycolytic activity, as indicated by the increased rate of fall in glucose. (c) Relative change in intracellular lactate concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, during inhibition of lactate efflux via MCT1 with 300 nm AR-C155858. (d) Rate of change in intracellular lactate concentration, after inhibition of lactate transport in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a robust increase in the rate of lactate production. (e) Relative change in the RNA level of GLUT1 and LDH1 in MCF-7 cells after three days under hypoxic conditions. Data are represented as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4559800&req=5

f5: Glycolysis and lactate production are augmented under hypoxic conditions.(a) Relative change in intracellular glucose concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, before and during inhibition of glucose uptake with 20 μM Cytochalasin B. (b) Rate of fall in intracellular glucose concentration, after inhibition of glucose uptake with Cytochalasin B in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a significant increase in glycolytic activity, as indicated by the increased rate of fall in glucose. (c) Relative change in intracellular lactate concentration in MCF-7 cells under normoxia (21% O2, black trace) and hypoxia (1% O2, blue trace), respectively, during inhibition of lactate efflux via MCT1 with 300 nm AR-C155858. (d) Rate of change in intracellular lactate concentration, after inhibition of lactate transport in MCF-7 cells under normoxia (21% O2, light grey bar) and hypoxia (1% O2, blue bar), respectively. Hypoxia leads to a robust increase in the rate of lactate production. (e) Relative change in the RNA level of GLUT1 and LDH1 in MCF-7 cells after three days under hypoxic conditions. Data are represented as mean ± SEM.
Mentions: To investigate the significance of CAIX-mediated facilitation of lactate/H+ transport capacity for cancer cell metabolism and propagation, we determined changes in glycolysis and lactate production during hypoxia on the single cell level. We measured changes in intracellular glucose and lactate concentration in individual MCF-7 breast cancer cells, incubated three days under normoxic or hypoxic conditions, with FLIP12glu-700 μΔ6 and Laconic, respectively. Under hypoxic conditions, the glycolytic rate increased to 170%, as measured by the rate of decrease in intracellular glucose during inhibition of glucose import by 20 μM Cytochalasin B (Fig. 5a,b; ref. 34.)Lactate production increased to 162% under these conditions, as measured by the rate of increase in intracellular lactate during inhibition of lactate efflux with 300 nm AR-C155858 (Fig. 5c,d; ref. 35). Furthermore the mRNA levels of the glucose transporter GLUT1 and lactate dehydrogenase LDH-1, two markers for glycolytic metabolism, increased under hypoxic conditions (Fig. 5e).

Bottom Line: Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction.Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1.Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

View Article: PubMed Central - PubMed

Affiliation: Division of Zoology/Membrane Transport, FB Biologie, TU Kaiserslautern, P.O. Box 3049, D-67653 Kaiserslautern, Germany.

ABSTRACT
The most aggressive tumour cells, which often reside in hypoxic environments, rely on glycolysis for energy production. Thereby they release vast amounts of lactate and protons via monocarboxylate transporters (MCTs), which exacerbates extracellular acidification and supports the formation of a hostile environment. We have studied the mechanisms of regulated lactate transport in MCF-7 human breast cancer cells. Under hypoxia, expression of MCT1 and MCT4 remained unchanged, while expression of carbonic anhydrase IX (CAIX) was greatly enhanced. Our results show that CAIX augments MCT1 transport activity by a non-catalytic interaction. Mutation studies in Xenopus oocytes indicate that CAIX, via its intramolecular H(+)-shuttle His200, functions as a "proton-collecting/distributing antenna" to facilitate rapid lactate flux via MCT1. Knockdown of CAIX significantly reduced proliferation of cancer cells, suggesting that rapid efflux of lactate and H(+), as enhanced by CAIX, contributes to cancer cell survival under hypoxic conditions.

No MeSH data available.


Related in: MedlinePlus