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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 the level of HIF-1α. HIF-1α protein level was increased in hypoxic HeLa cells cultured in the presence of 20 mM carnosine for 48 h as demonstrated by western blot (A) and immunofluorescence (scale bar, 10 μm) (B). (C) Carnosine treatment increased expression of HIF-1α mRNA and its target genes VEGF and GLUT-1 under hypoxic conditions. Concurrently, the mRNA level of VBP1, a protein that binds to VHL and is involved in HIF-1α degradation, was decreased. (D) Chromatin immunoprecipitation assay performed under the same conditions demonstrated that HIF-1α bound to hypoxia-responsive elements in the promoter region of VEGF and CA9 genes.
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Figure 3: Effect of carnosine on the level of HIF-1α. HIF-1α protein level was increased in hypoxic HeLa cells cultured in the presence of 20 mM carnosine for 48 h as demonstrated by western blot (A) and immunofluorescence (scale bar, 10 μm) (B). (C) Carnosine treatment increased expression of HIF-1α mRNA and its target genes VEGF and GLUT-1 under hypoxic conditions. Concurrently, the mRNA level of VBP1, a protein that binds to VHL and is involved in HIF-1α degradation, was decreased. (D) Chromatin immunoprecipitation assay performed under the same conditions demonstrated that HIF-1α bound to hypoxia-responsive elements in the promoter region of VEGF and CA9 genes.

Mentions: Because transcription of CA IX is activated by HIF-1α, we tested whether carnosine influenced HIF-1α protein and mRNA levels in HeLa cells. HeLa cells were cultured in hypoxic conditions for 48 h with or without 20 mM carnosine. Western blot analysis showed a significant increase in HIF-1α signal in cells treated with carnosine compared with controls (Figure 3A). This finding was supported by immunofluorescent staining of HIF-1α, which showed a stronger HIF-1α signal in the nuclei of treated cells (Figure 3B). Data from qPCR analysis confirmed an increased level of HIF-1α mRNA after carnosine treatment under hypoxia compared with the untreated control (Figure 3C). The activity of HIF-1α was demonstrated by the increase in mRNA expression of the HIF-1α targets vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1) after carnosine treatment in hypoxia (Figure 3C). Moreover, ChIP analysis showed a moderate increase in binding of HIF-1α to the HRE in both CA9 and VEGF promoters (Figure 3D). Interestingly, the level of VBP1 mRNA decreased after carnosine treatment compared with the control (Figure 3C), indicating reduced degradation and increased stabilization of HIF-1α protein.


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 HIF-1α. HIF-1α protein level was increased in hypoxic HeLa cells cultured in the presence of 20 mM carnosine for 48 h as demonstrated by western blot (A) and immunofluorescence (scale bar, 10 μm) (B). (C) Carnosine treatment increased expression of HIF-1α mRNA and its target genes VEGF and GLUT-1 under hypoxic conditions. Concurrently, the mRNA level of VBP1, a protein that binds to VHL and is involved in HIF-1α degradation, was decreased. (D) Chromatin immunoprecipitation assay performed under the same conditions demonstrated that HIF-1α bound to hypoxia-responsive elements in the promoter region of VEGF and CA9 genes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Effect of carnosine on the level of HIF-1α. HIF-1α protein level was increased in hypoxic HeLa cells cultured in the presence of 20 mM carnosine for 48 h as demonstrated by western blot (A) and immunofluorescence (scale bar, 10 μm) (B). (C) Carnosine treatment increased expression of HIF-1α mRNA and its target genes VEGF and GLUT-1 under hypoxic conditions. Concurrently, the mRNA level of VBP1, a protein that binds to VHL and is involved in HIF-1α degradation, was decreased. (D) Chromatin immunoprecipitation assay performed under the same conditions demonstrated that HIF-1α bound to hypoxia-responsive elements in the promoter region of VEGF and CA9 genes.
Mentions: Because transcription of CA IX is activated by HIF-1α, we tested whether carnosine influenced HIF-1α protein and mRNA levels in HeLa cells. HeLa cells were cultured in hypoxic conditions for 48 h with or without 20 mM carnosine. Western blot analysis showed a significant increase in HIF-1α signal in cells treated with carnosine compared with controls (Figure 3A). This finding was supported by immunofluorescent staining of HIF-1α, which showed a stronger HIF-1α signal in the nuclei of treated cells (Figure 3B). Data from qPCR analysis confirmed an increased level of HIF-1α mRNA after carnosine treatment under hypoxia compared with the untreated control (Figure 3C). The activity of HIF-1α was demonstrated by the increase in mRNA expression of the HIF-1α targets vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT-1) after carnosine treatment in hypoxia (Figure 3C). Moreover, ChIP analysis showed a moderate increase in binding of HIF-1α to the HRE in both CA9 and VEGF promoters (Figure 3D). Interestingly, the level of VBP1 mRNA decreased after carnosine treatment compared with the control (Figure 3C), indicating reduced degradation and increased stabilization of HIF-1α protein.

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