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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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Effect of carnosine on spheroid size and cell viability. (A) Images of spheroids cultured in the presence and absence of 20 mM carnosine. Carnosine was added after spheroids had already formed (upper image) or before spheroid formation (lower image). The images show the same time points of spheroid growth, although the duration of carnosine treatment differs as indicated, depending whether carnosine was added before or after spheroid formation. pH values represent measured pH of media collected from all respective spheroid samples (N > 30) at the end of the experiment. (B) In both experimental conditions, the diameters of carnosine-treated spheroids were significantly smaller than those of controls (t-test, **p < 0.01). (C) Flow cytometry showed that treatment with different concentrations of carnosine (5–40 mM) decreased cell viability in hypoxic conditions in a dose-dependent manner (t-test, *p < 0.05, **p < 0.01). Numbers in the columns give the ratio for the viability of a carnosine-treated sample and the respective hypoxic control (set as 100%). The ratio of the viability of hypoxic and normoxic controls was 96%. (D) Cultivation of HeLa spheroids in the presence of 20 mM carnosine significantly reduced cell viability by approximately 50% compared with the control group (t-test, **p < 0.01).
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Figure 5: Effect of carnosine on spheroid size and cell viability. (A) Images of spheroids cultured in the presence and absence of 20 mM carnosine. Carnosine was added after spheroids had already formed (upper image) or before spheroid formation (lower image). The images show the same time points of spheroid growth, although the duration of carnosine treatment differs as indicated, depending whether carnosine was added before or after spheroid formation. pH values represent measured pH of media collected from all respective spheroid samples (N > 30) at the end of the experiment. (B) In both experimental conditions, the diameters of carnosine-treated spheroids were significantly smaller than those of controls (t-test, **p < 0.01). (C) Flow cytometry showed that treatment with different concentrations of carnosine (5–40 mM) decreased cell viability in hypoxic conditions in a dose-dependent manner (t-test, *p < 0.05, **p < 0.01). Numbers in the columns give the ratio for the viability of a carnosine-treated sample and the respective hypoxic control (set as 100%). The ratio of the viability of hypoxic and normoxic controls was 96%. (D) Cultivation of HeLa spheroids in the presence of 20 mM carnosine significantly reduced cell viability by approximately 50% compared with the control group (t-test, **p < 0.01).

Mentions: To confirm the effect of carnosine in a physiologically more relevant three-dimensional (3D) environment, we treated spheroids formed by HeLa cells with carnosine added to the culture medium only after the spheroids had already formed, or with carnosine present during the period of spheroid formation. Both experimental groups formed spheroids, indicating that spheroid formation was not significantly affected by carnosine. At the end of the experiment the carnosine-treated spheroids in both groups had a significantly smaller (almost 50% smaller) diameter than the controls; moreover, the extracellular pH of the treated groups was higher in the treated cultures than in the controls (Figure 5A, 5B).Data from flow cytometric analysis showed that carnosine treatment of a two-dimensional monolayer culture decreased the viability of hypoxic cells in a dose-dependent manner: 5 mM carnosine decreased HeLa cells viability only slightly, 10 mM carnosine by approximately 10%, and 20 mM by approximately 15% (Figure 5C). In comparison, the viability of HeLa cells in normoxic conditions remained relatively constant in the presence of different concentrations of carnosine (data not shown). In 3D culture, where hypoxia develops in the center of spheroids, we observed a marked decrease in viability of HeLa spheroids of 50% after treatment with 20 mM carnosine compared with the controls (Figure 5D).


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 spheroid size and cell viability. (A) Images of spheroids cultured in the presence and absence of 20 mM carnosine. Carnosine was added after spheroids had already formed (upper image) or before spheroid formation (lower image). The images show the same time points of spheroid growth, although the duration of carnosine treatment differs as indicated, depending whether carnosine was added before or after spheroid formation. pH values represent measured pH of media collected from all respective spheroid samples (N > 30) at the end of the experiment. (B) In both experimental conditions, the diameters of carnosine-treated spheroids were significantly smaller than those of controls (t-test, **p < 0.01). (C) Flow cytometry showed that treatment with different concentrations of carnosine (5–40 mM) decreased cell viability in hypoxic conditions in a dose-dependent manner (t-test, *p < 0.05, **p < 0.01). Numbers in the columns give the ratio for the viability of a carnosine-treated sample and the respective hypoxic control (set as 100%). The ratio of the viability of hypoxic and normoxic controls was 96%. (D) Cultivation of HeLa spheroids in the presence of 20 mM carnosine significantly reduced cell viability by approximately 50% compared with the control group (t-test, **p < 0.01).
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Related In: Results  -  Collection

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Figure 5: Effect of carnosine on spheroid size and cell viability. (A) Images of spheroids cultured in the presence and absence of 20 mM carnosine. Carnosine was added after spheroids had already formed (upper image) or before spheroid formation (lower image). The images show the same time points of spheroid growth, although the duration of carnosine treatment differs as indicated, depending whether carnosine was added before or after spheroid formation. pH values represent measured pH of media collected from all respective spheroid samples (N > 30) at the end of the experiment. (B) In both experimental conditions, the diameters of carnosine-treated spheroids were significantly smaller than those of controls (t-test, **p < 0.01). (C) Flow cytometry showed that treatment with different concentrations of carnosine (5–40 mM) decreased cell viability in hypoxic conditions in a dose-dependent manner (t-test, *p < 0.05, **p < 0.01). Numbers in the columns give the ratio for the viability of a carnosine-treated sample and the respective hypoxic control (set as 100%). The ratio of the viability of hypoxic and normoxic controls was 96%. (D) Cultivation of HeLa spheroids in the presence of 20 mM carnosine significantly reduced cell viability by approximately 50% compared with the control group (t-test, **p < 0.01).
Mentions: To confirm the effect of carnosine in a physiologically more relevant three-dimensional (3D) environment, we treated spheroids formed by HeLa cells with carnosine added to the culture medium only after the spheroids had already formed, or with carnosine present during the period of spheroid formation. Both experimental groups formed spheroids, indicating that spheroid formation was not significantly affected by carnosine. At the end of the experiment the carnosine-treated spheroids in both groups had a significantly smaller (almost 50% smaller) diameter than the controls; moreover, the extracellular pH of the treated groups was higher in the treated cultures than in the controls (Figure 5A, 5B).Data from flow cytometric analysis showed that carnosine treatment of a two-dimensional monolayer culture decreased the viability of hypoxic cells in a dose-dependent manner: 5 mM carnosine decreased HeLa cells viability only slightly, 10 mM carnosine by approximately 10%, and 20 mM by approximately 15% (Figure 5C). In comparison, the viability of HeLa cells in normoxic conditions remained relatively constant in the presence of different concentrations of carnosine (data not shown). In 3D culture, where hypoxia develops in the center of spheroids, we observed a marked decrease in viability of HeLa spheroids of 50% after treatment with 20 mM carnosine compared with the controls (Figure 5D).

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