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The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

ABSTRACT: We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of approximately 300 microm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pH(i) (approximately 6.3) and reduced pH(e) (approximately 6.9) at their core, associated with a diminishing gradient of acidity extending out to the periphery. With CA9 expression, core intracellular acidity was less prominent (pH(i) = approximately 6.6), whereas extracellular acidity was enhanced (pH(e) = approximately 6.6), so that radial pH(i) gradients were smaller and radial pH(e) gradients were larger. These effects were reversed by eliminating CA9 activity with membrane-impermeant CA inhibitors. The observation that CA9 activity reversibly reduces pH(e) indicates the enzyme is facilitating CO(2) excretion from cells (by converting vented CO(2) to extracellular H(+)), rather than facilitating membrane H(+) transport (such as H(+) associated with metabolically generated lactic acid). This latter process requires titration of exported H(+) ions with extracellular HCO(3)(-), which would reduce rather than increase extracellular acidity. In a multicellular structure, the net effect of CA9 on pH(e) will depend on the cellular CO(2)/lactic acid emission ratio (set by local oxygenation and membrane HCO(3)(-) uptake). Our results suggest that CO(2)-producing tumors may express CA9 to facilitate CO(2) excretion, thus raising pH(i) and reducing pH(e), which promotes tumor proliferation and survival. The results suggest a possible basis for attenuating tumor development through inhibiting CA9 activity.

SUBMITTER: Swietach P

PROVIDER: S-EPMC2740455 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

Swietach Pawel P Patiar Shalini S Supuran Claudiu T CT Harris Adrian L AL Vaughan-Jones Richard D RD

The Journal of biological chemistry 20090519 30

We have studied the role of carbonic anhydrase 9 (CA9), a cancer-associated extracellular isoform of the enzyme carbonic anhydrase in multicellular spheroid growths (radius of approximately 300 microm) of human colon carcinoma HCT116 cells. Spheroids were transfected with CA9 (or empty vector) and imaged confocally (using fluorescent dyes) for both intracellular pH (pH(i)) and pH in the restricted extracellular spaces (pH(e)). With no CA9 expression, spheroids developed very low pH(i) (approxima ...[more]

PMID: 19458084

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Project description: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 (?-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-1? and CA IX and/or altered CA IX function.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-1? 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.Carnosine increased the expression levels of HIF-1? 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.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.

Project description:Silk is a protein of interest to both biological and industrial sciences. The silkworm, Bombyx mori, forms this protein into strong threads starting from soluble silk proteins using a number of biochemical and physical cues to allow the transition from liquid to fibrous silk. A pH gradient has been measured along the gland, but the methodology employed was not able to precisely determine the pH at specific regions of interest in the silk gland. Furthermore, the physiological mechanisms responsible for the generation of this pH gradient are unknown. In this study, concentric ion selective microelectrodes were used to determine the luminal pH of B. mori silk glands. A gradient from pH 8.2 to 7.2 was measured in the posterior silk gland, with a pH 7 throughout the middle silk gland, and a gradient from pH 6.8 to 6.2 in the beginning of the anterior silk gland where silk processing into fibers occurs. The small diameter of the most anterior region of the anterior silk gland prevented microelectrode access in this region. Using a histochemical method, the presence of active carbonic anhydrase was identified in the funnel and anterior silk gland of fifth instar larvae. The observed pH gradient collapsed upon addition of the carbonic anhydrase inhibitor methazolamide, confirming an essential role for this enzyme in pH regulation in the B. mori silk gland. Plastic embedding of whole silk glands allowed clear visualization of the morphology, including the identification of four distinct epithelial cell types in the gland and allowed correlations between silk gland morphology and silk stages of assembly related to the pH gradient. B. mori silk glands have four different epithelial cell types, one of which produces carbonic anhydrase. Carbonic anhydrase is necessary for the mechanism that generates an intraluminal pH gradient, which likely regulates the assembly of silk proteins and then the formation of fibers from soluble silk proteins. These new insights into native silk formation may lead to a more efficient production of artificial or regenerated silkworm silk fibers.

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The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

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The role of carbonic anhydrase 9 in regulating extracellular and intracellular ph in three-dimensional tumor cell growths.

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