Abstract
BackgroundCarbonic 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.MethodsThe 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.ResultsCarnosine 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.ConclusionsOur 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.
Highlights
Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors
Because kinetic and X-ray crystallographic studies suggest that carnosine is a potent activator of the carbonic anhydrase isoforms hCA I, II, and IV [24] and the studies described above indicate that carnosine affects the hypoxia-inducible factor (HIF)-1 signaling pathway, we initially examined whether CA IX is involved in the antitumor activity of carnosine
Carnosine reduces the extracellular acidosis linked to catalytic activity of CA IX in hypoxia and inhibits the growth of spheroids and tumor xenografts
Summary
Carbonic anhydrase IX (CA IX) is a transmembrane enzyme that is present in many types of solid tumors. Stabilization of hypoxia-inducible factor 1 (HIF-1) as an adaptive response to hypoxic conditions in tissues results in transcriptional activation of many genes that play an important role in cancer-related processes, such as angiogenesis, cell survival, glucose metabolism, and cell invasion. Carnosine plays a role as a physiologic pH buffering substance and antioxidant [4] It induces variable effects on the cardiovascular system, including down-regulation of blood pressure [5,6], inhibition of glycosylated lowdensity lipoprotein formation [7], and inhibition of angiotensin-converting enzyme activity [8]. It acts as an anti-aging agent [9]. The mechanism of its action in tumor cells remains unclear
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