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Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts.

Ditte Z, Ditte P, Labudova M, Simko V, Iuliano F, Zatovicova M, Csaderova L, Pastorekova S, Pastorek J - BMC Cancer (2014)

Bottom Line: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis.This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 05, Slovak Republic. virupast@savba.sk.

ABSTRACT

Background: Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function.

Methods: The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.

Results: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.

Conclusions: Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.

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Related in: MedlinePlus

Effect of carnosine on the level of CA IX protein. HeLa cells were cultured in hypoxic conditions for 48 h in the presence of 20 mM carnosine. Carnosine induced a slight increase in CA IX protein level in hypoxic cells as shown by western blot analysis (A) and immunofluorescence (scale bar 20 μm) (B). This result was confirmed by flow cytometric analysis using M75 antibody against CA IX (C). After treatment with 20 mM carnosine, the level of membrane-localized CA IX increased by 48% compared with the control. Other concentrations of carnosine did not have a comparable effect on CA IX protein level. Differences between the untreated control and carnosine-treated samples were evaluated by a t-test (*p < 0.05).
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Figure 2: Effect of carnosine on the level of CA IX protein. HeLa cells were cultured in hypoxic conditions for 48 h in the presence of 20 mM carnosine. Carnosine induced a slight increase in CA IX protein level in hypoxic cells as shown by western blot analysis (A) and immunofluorescence (scale bar 20 μm) (B). This result was confirmed by flow cytometric analysis using M75 antibody against CA IX (C). After treatment with 20 mM carnosine, the level of membrane-localized CA IX increased by 48% compared with the control. Other concentrations of carnosine did not have a comparable effect on CA IX protein level. Differences between the untreated control and carnosine-treated samples were evaluated by a t-test (*p < 0.05).

Mentions: To determine whether the carnosine-mediated reduction in extracellular acidification of CA IX-positive cells is related to CA IX protein level, we cultivated HeLa cells in hypoxic conditions and used our in-house anti-CA IX antibody M75 to measure CA IX protein levels. The level of CA IX protein increased after carnosine treatment (Figure 2A). This result was confirmed by immunofluorescent staining of CA IX (Figure 2B) and by flow cytometry analysis, which showed that 20 mM carnosine treatment increased the levels of surface CA IX in HeLa cells under hypoxia (Figure 2C). Carnosine did not change the degree of phosphorylation at Thr443, suggesting that it has no effect on activation of CA IX through phosphorylation by PKA (Additional file 2).


Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts.

Ditte Z, Ditte P, Labudova M, Simko V, Iuliano F, Zatovicova M, Csaderova L, Pastorekova S, Pastorek J - BMC Cancer (2014)

Effect of carnosine on the level of CA IX protein. HeLa cells were cultured in hypoxic conditions for 48 h in the presence of 20 mM carnosine. Carnosine induced a slight increase in CA IX protein level in hypoxic cells as shown by western blot analysis (A) and immunofluorescence (scale bar 20 μm) (B). This result was confirmed by flow cytometric analysis using M75 antibody against CA IX (C). After treatment with 20 mM carnosine, the level of membrane-localized CA IX increased by 48% compared with the control. Other concentrations of carnosine did not have a comparable effect on CA IX protein level. Differences between the untreated control and carnosine-treated samples were evaluated by a t-test (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4061103&req=5

Figure 2: Effect of carnosine on the level of CA IX protein. HeLa cells were cultured in hypoxic conditions for 48 h in the presence of 20 mM carnosine. Carnosine induced a slight increase in CA IX protein level in hypoxic cells as shown by western blot analysis (A) and immunofluorescence (scale bar 20 μm) (B). This result was confirmed by flow cytometric analysis using M75 antibody against CA IX (C). After treatment with 20 mM carnosine, the level of membrane-localized CA IX increased by 48% compared with the control. Other concentrations of carnosine did not have a comparable effect on CA IX protein level. Differences between the untreated control and carnosine-treated samples were evaluated by a t-test (*p < 0.05).
Mentions: To determine whether the carnosine-mediated reduction in extracellular acidification of CA IX-positive cells is related to CA IX protein level, we cultivated HeLa cells in hypoxic conditions and used our in-house anti-CA IX antibody M75 to measure CA IX protein levels. The level of CA IX protein increased after carnosine treatment (Figure 2A). This result was confirmed by immunofluorescent staining of CA IX (Figure 2B) and by flow cytometry analysis, which showed that 20 mM carnosine treatment increased the levels of surface CA IX in HeLa cells under hypoxia (Figure 2C). Carnosine did not change the degree of phosphorylation at Thr443, suggesting that it has no effect on activation of CA IX through phosphorylation by PKA (Additional file 2).

Bottom Line: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis.This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Molecular Medicine, Institute of Virology, Slovak Academy of Sciences, Dubravska cesta 9, Bratislava 845 05, Slovak Republic. virupast@savba.sk.

ABSTRACT

Background: Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Expression of CA IX is driven predominantly by the hypoxia-inducible factor (HIF) pathway and helps to maintain intracellular pH homeostasis under hypoxic conditions, resulting in acidification of the tumor microenvironment. Carnosine (β-alanyl-L-histidine) is an anti-tumorigenic agent that inhibits the proliferation of cancer cells. In this study, we investigated the role of CA IX in carnosine-mediated antitumor activity and whether the underlying mechanism involves transcriptional and translational modulation of HIF-1α and CA IX and/or altered CA IX function.

Methods: The effect of carnosine was studied using two-dimensional cell monolayers of several cell lines with endogenous CA IX expression as well as Madin Darby canine kidney transfectants, three-dimensional HeLa spheroids, and an in vivo model of HeLa xenografts in nude mice. mRNA and protein expression and protein localization were analyzed by real-time PCR, western blot analysis, and immunofluorescence staining, respectively. Cell viability was measured by a flow cytometric assay. Expression of HIF-1α and CA IX in tumors was assessed by immunohistochemical staining. Real-time measurement of pH was performed using a sensor dish reader. Binding of CA IX to specific antibodies and metabolon partners was investigated by competitive ELISA and proximity ligation assays, respectively.

Results: Carnosine increased the expression levels of HIF-1α and HIF targets and increased the extracellular pH, suggesting an inhibitory effect on CA IX-mediated acidosis. Moreover, carnosine significantly inhibited the growth of three-dimensional spheroids and tumor xenografts compared with untreated controls. Competitive ELISA showed that carnosine disrupted binding between CA IX and antibodies specific for its catalytic domain. This finding was supported by reduced formation of the functional metabolon of CA IX and anion exchanger 2 in the presence of carnosine.

Conclusions: Our results indicate that interaction of carnosine with CA IX leads to conformational changes of CA IX and impaired formation of its metabolon, which in turn disrupts CA IX function. These findings suggest that carnosine could be a promising anticancer drug through its ability to attenuate the activity of CA IX.

Show MeSH
Related in: MedlinePlus