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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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Carnosine decreases acidification of the growth media of different cell lines. (A) HeLa cells were incubated for 48 h in hypoxic conditions in the presence of different concentrations of carnosine (5–40 mM) and the extracellular pH was measured by SDR in real time. Carnosine treatment markedly decreased the acidification of growth media in a dose-dependent manner. **denotes p < 0.01 for the comparison between samples treated with different carnosine concentrations and the untreated control. (B) SiHa, HeLa, HT-29, MDCK-CA IX, and MDCK-mock cells were exposed to hypoxia for 48 h in the presence of 20 mM carnosine. The columns in the graph represent differences in the endpoint pH values of carnosine-treated cells and their respective untreated controls. Carnosine caused considerable alkalization of growth media in all studied cell lines, except for MDCK mock cells in which the effect of carnosine was noticeably lower. Differences between endpoint pH values of the untreated control and carnosine-treated cells were evaluated by a t-test (*p < 0.05, **p < 0.01).
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Figure 1: Carnosine decreases acidification of the growth media of different cell lines. (A) HeLa cells were incubated for 48 h in hypoxic conditions in the presence of different concentrations of carnosine (5–40 mM) and the extracellular pH was measured by SDR in real time. Carnosine treatment markedly decreased the acidification of growth media in a dose-dependent manner. **denotes p < 0.01 for the comparison between samples treated with different carnosine concentrations and the untreated control. (B) SiHa, HeLa, HT-29, MDCK-CA IX, and MDCK-mock cells were exposed to hypoxia for 48 h in the presence of 20 mM carnosine. The columns in the graph represent differences in the endpoint pH values of carnosine-treated cells and their respective untreated controls. Carnosine caused considerable alkalization of growth media in all studied cell lines, except for MDCK mock cells in which the effect of carnosine was noticeably lower. Differences between endpoint pH values of the untreated control and carnosine-treated cells were evaluated by a t-test (*p < 0.05, **p < 0.01).

Mentions: Cultivation of HeLa cells under hypoxia for 48 h in the presence of carnosine (5–40 mM) resulted in reduced acidification of the extracellular environment in a dose-dependent manner (Figure 1A). The effect of carnosine on HeLa cells in normoxic conditions was substantially smaller (data not shown). Because of its physiologic relevance, a carnosine concentration of 20 mM was selected for further tests on different cancer cell lines (SiHa, HeLa, HT-29). Incubation with carnosine markedly reduced the acidification of growth media in hypoxic conditions for all cell lines studied (Figure 1B, Additional file 1). We also observed a carnosine-mediated decrease in acidification in MDCK cells transfected with CA IX, whereas the effect of carnosine on their mock-transfected counterparts was considerably smaller.


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)

Carnosine decreases acidification of the growth media of different cell lines. (A) HeLa cells were incubated for 48 h in hypoxic conditions in the presence of different concentrations of carnosine (5–40 mM) and the extracellular pH was measured by SDR in real time. Carnosine treatment markedly decreased the acidification of growth media in a dose-dependent manner. **denotes p < 0.01 for the comparison between samples treated with different carnosine concentrations and the untreated control. (B) SiHa, HeLa, HT-29, MDCK-CA IX, and MDCK-mock cells were exposed to hypoxia for 48 h in the presence of 20 mM carnosine. The columns in the graph represent differences in the endpoint pH values of carnosine-treated cells and their respective untreated controls. Carnosine caused considerable alkalization of growth media in all studied cell lines, except for MDCK mock cells in which the effect of carnosine was noticeably lower. Differences between endpoint pH values of the untreated control and carnosine-treated cells were evaluated by a t-test (*p < 0.05, **p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Carnosine decreases acidification of the growth media of different cell lines. (A) HeLa cells were incubated for 48 h in hypoxic conditions in the presence of different concentrations of carnosine (5–40 mM) and the extracellular pH was measured by SDR in real time. Carnosine treatment markedly decreased the acidification of growth media in a dose-dependent manner. **denotes p < 0.01 for the comparison between samples treated with different carnosine concentrations and the untreated control. (B) SiHa, HeLa, HT-29, MDCK-CA IX, and MDCK-mock cells were exposed to hypoxia for 48 h in the presence of 20 mM carnosine. The columns in the graph represent differences in the endpoint pH values of carnosine-treated cells and their respective untreated controls. Carnosine caused considerable alkalization of growth media in all studied cell lines, except for MDCK mock cells in which the effect of carnosine was noticeably lower. Differences between endpoint pH values of the untreated control and carnosine-treated cells were evaluated by a t-test (*p < 0.05, **p < 0.01).
Mentions: Cultivation of HeLa cells under hypoxia for 48 h in the presence of carnosine (5–40 mM) resulted in reduced acidification of the extracellular environment in a dose-dependent manner (Figure 1A). The effect of carnosine on HeLa cells in normoxic conditions was substantially smaller (data not shown). Because of its physiologic relevance, a carnosine concentration of 20 mM was selected for further tests on different cancer cell lines (SiHa, HeLa, HT-29). Incubation with carnosine markedly reduced the acidification of growth media in hypoxic conditions for all cell lines studied (Figure 1B, Additional file 1). We also observed a carnosine-mediated decrease in acidification in MDCK cells transfected with CA IX, whereas the effect of carnosine on their mock-transfected counterparts was considerably smaller.

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