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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

Lactate flux in MCF-7 cancer cells is augmented under hypoxic conditions.(a) Relative change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 1 and 3 mM lactate, measured with Laconic. (b) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application of 1 and 3 mM lactate. Hypoxia induces a significant increase in lactate flux. (c) Change in intracellular pH (pHi) in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 3 and 10 mM lactate in the absence and presence of the MCT1 inhibitor AR-C155858. (d) Rate of change in pHi in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application and removal, of 3 and 10 mM lactate, respectively. Rate of lactate-induced proton flux is augmented under hypoxic conditions. AR-C155858 fully inhibits proton flux. Data are represented as mean ± SEM.
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f1: Lactate flux in MCF-7 cancer cells is augmented under hypoxic conditions.(a) Relative change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 1 and 3 mM lactate, measured with Laconic. (b) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application of 1 and 3 mM lactate. Hypoxia induces a significant increase in lactate flux. (c) Change in intracellular pH (pHi) in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 3 and 10 mM lactate in the absence and presence of the MCT1 inhibitor AR-C155858. (d) Rate of change in pHi in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application and removal, of 3 and 10 mM lactate, respectively. Rate of lactate-induced proton flux is augmented under hypoxic conditions. AR-C155858 fully inhibits proton flux. Data are represented as mean ± SEM.

Mentions: Hypoxia triggers a glycolytic switch in tumour tissues, resulting in increased production of lactate and H+. To test whether lactate/H+ transport capacity is increased by hypoxia, we measured lactate flux in MCF-7 cells during application of 1 and 3 mM lactate under normoxic and hypoxic conditions by single-cell lactate imaging with the FRET-based lactate nanosensor Laconic (Fig. 1a). Indeed, the rate of lactate rise increased to 225% at 1 mM and 140% at 3 mM lactate under hypoxic conditions (Fig. 1b). Since Laconic has a high affinity for lactate, the sensor is well suited to reliably measure the rate of lactate uptake, but not the rate of lactate efflux (due to the high time constant of lactate release from the sensor the rate of efflux may be underestimated). To determine efflux capacity, we measured changes in intracellular pH in MCF-7 cells during application and removal of 3 and 10 mM lactate under normoxic and hypoxic conditions by pH imaging (Fig. 1c). Under hypoxia, the lactate-induced rate of change in pHi increased both during application and removal of lactate, suggesting a hypoxia-dependent increase in lactate/H+ influx and efflux (Fig. 1d). Application of 3 and 10 mM lactate in the presence of the MCT1 inhibitor AR-C155858 (300 nM) led to no change in pHi neither in normoxic, nor in hypoxic cells (Fig. 1c,d). Since the cells constantly produce lactate and H+, inhibition of lactate/H+ efflux leads to a constant intracellular acidification (Fig. 1c). Therefore ΔpHi/Δt measured shortly after application of AR-C155858 was subtracted from ΔpHi/Δt measured during application of lactate in the presence of the inhibitor.


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)

Lactate flux in MCF-7 cancer cells is augmented under hypoxic conditions.(a) Relative change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 1 and 3 mM lactate, measured with Laconic. (b) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application of 1 and 3 mM lactate. Hypoxia induces a significant increase in lactate flux. (c) Change in intracellular pH (pHi) in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 3 and 10 mM lactate in the absence and presence of the MCT1 inhibitor AR-C155858. (d) Rate of change in pHi in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application and removal, of 3 and 10 mM lactate, respectively. Rate of lactate-induced proton flux is augmented under hypoxic conditions. AR-C155858 fully inhibits proton flux. Data are represented as mean ± SEM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4559800&req=5

f1: Lactate flux in MCF-7 cancer cells is augmented under hypoxic conditions.(a) Relative change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 1 and 3 mM lactate, measured with Laconic. (b) Rate of change in intracellular lactate concentration in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application of 1 and 3 mM lactate. Hypoxia induces a significant increase in lactate flux. (c) Change in intracellular pH (pHi) in MCF-7 cells under normoxic (21% O2, black trace) and hypoxic (1% O2, blue trace) conditions, respectively, as induced by application of 3 and 10 mM lactate in the absence and presence of the MCT1 inhibitor AR-C155858. (d) Rate of change in pHi in MCF-7 cells under normoxic (21% O2) and hypoxic (1% O2) conditions, respectively, as induced by application and removal, of 3 and 10 mM lactate, respectively. Rate of lactate-induced proton flux is augmented under hypoxic conditions. AR-C155858 fully inhibits proton flux. Data are represented as mean ± SEM.
Mentions: Hypoxia triggers a glycolytic switch in tumour tissues, resulting in increased production of lactate and H+. To test whether lactate/H+ transport capacity is increased by hypoxia, we measured lactate flux in MCF-7 cells during application of 1 and 3 mM lactate under normoxic and hypoxic conditions by single-cell lactate imaging with the FRET-based lactate nanosensor Laconic (Fig. 1a). Indeed, the rate of lactate rise increased to 225% at 1 mM and 140% at 3 mM lactate under hypoxic conditions (Fig. 1b). Since Laconic has a high affinity for lactate, the sensor is well suited to reliably measure the rate of lactate uptake, but not the rate of lactate efflux (due to the high time constant of lactate release from the sensor the rate of efflux may be underestimated). To determine efflux capacity, we measured changes in intracellular pH in MCF-7 cells during application and removal of 3 and 10 mM lactate under normoxic and hypoxic conditions by pH imaging (Fig. 1c). Under hypoxia, the lactate-induced rate of change in pHi increased both during application and removal of lactate, suggesting a hypoxia-dependent increase in lactate/H+ influx and efflux (Fig. 1d). Application of 3 and 10 mM lactate in the presence of the MCT1 inhibitor AR-C155858 (300 nM) led to no change in pHi neither in normoxic, nor in hypoxic cells (Fig. 1c,d). Since the cells constantly produce lactate and H+, inhibition of lactate/H+ efflux leads to a constant intracellular acidification (Fig. 1c). Therefore ΔpHi/Δt measured shortly after application of AR-C155858 was subtracted from ΔpHi/Δt measured during application of lactate in the presence of the inhibitor.

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