Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of carnosine in a mouse xenograft model. (A) Graph showing slower growth of tumors in the group treated with carnosine. Carnosine treatment began on the 14th day after subcutaneous implantation of HeLa cells, and tumor size was measured using a caliper during treatment. (B) There was a significant difference in the weight of tumors from treated and control groups (t-test, **p < 0.01). (C) Immunohistochemical staining of tumor tissue sections demonstrated enhanced expression of HIF-1α and CA IX proteins in the carnosine-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of carnosine in a mouse xenograft model. (A) Graph showing slower growth of tumors in the group treated with carnosine. Carnosine treatment began on the 14th day after subcutaneous implantation of HeLa cells, and tumor size was measured using a caliper during treatment. (B) There was a significant difference in the weight of tumors from treated and control groups (t-test, **p < 0.01). (C) Immunohistochemical staining of tumor tissue sections demonstrated enhanced expression of HIF-1α and CA IX proteins in the carnosine-treated group.

Mentions: Tumor growth was visible 7 days after subcutaneous implantation of HeLa cells in all animals. On the 14th day of the experiment, we separated the mice into two groups and started subcutaneous administration of carnosine solution to animals in the carnosine group. At the same time, we commenced caliper measurement of the tumors. All animals had comparable-sized tumors at the start of carnosine treatment. Between the 21st and 28th day of the experiment we noticed faster growth of tumors in the control group compared with the carnosine-treated group, in which the average tumor size remained relatively constant. Although several tumors continued to grow in the carnosine-treated group, the rate of tumor growth was very slow, whereas the growth of some tumors stopped and several tumors even became smaller (Figure 6A). These observations were confirmed after the final examination, when we found a significant difference in the weight of tumors between the control and carnosine-treated groups (Figure 6B). Immunohistochemical staining of formalin-fixed, paraffin-embedded tumor tissues demonstrated enhanced intensity of HIF1-α and CA IX staining in the carnosine-treated group (Figure 6C). These results correspond with the higher level of both proteins observed in vitro.


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 in a mouse xenograft model. (A) Graph showing slower growth of tumors in the group treated with carnosine. Carnosine treatment began on the 14th day after subcutaneous implantation of HeLa cells, and tumor size was measured using a caliper during treatment. (B) There was a significant difference in the weight of tumors from treated and control groups (t-test, **p < 0.01). (C) Immunohistochemical staining of tumor tissue sections demonstrated enhanced expression of HIF-1α and CA IX proteins in the carnosine-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of carnosine in a mouse xenograft model. (A) Graph showing slower growth of tumors in the group treated with carnosine. Carnosine treatment began on the 14th day after subcutaneous implantation of HeLa cells, and tumor size was measured using a caliper during treatment. (B) There was a significant difference in the weight of tumors from treated and control groups (t-test, **p < 0.01). (C) Immunohistochemical staining of tumor tissue sections demonstrated enhanced expression of HIF-1α and CA IX proteins in the carnosine-treated group.
Mentions: Tumor growth was visible 7 days after subcutaneous implantation of HeLa cells in all animals. On the 14th day of the experiment, we separated the mice into two groups and started subcutaneous administration of carnosine solution to animals in the carnosine group. At the same time, we commenced caliper measurement of the tumors. All animals had comparable-sized tumors at the start of carnosine treatment. Between the 21st and 28th day of the experiment we noticed faster growth of tumors in the control group compared with the carnosine-treated group, in which the average tumor size remained relatively constant. Although several tumors continued to grow in the carnosine-treated group, the rate of tumor growth was very slow, whereas the growth of some tumors stopped and several tumors even became smaller (Figure 6A). These observations were confirmed after the final examination, when we found a significant difference in the weight of tumors between the control and carnosine-treated groups (Figure 6B). Immunohistochemical staining of formalin-fixed, paraffin-embedded tumor tissues demonstrated enhanced intensity of HIF1-α and CA IX staining in the carnosine-treated group (Figure 6C). These results correspond with the higher level of both proteins observed in vitro.

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