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. 2019 Dec;34(1):272-278.
doi: 10.1080/14756366.2018.1538980.

Prostate cancer cells and exosomes in acidic condition show increased carbonic anhydrase IX expression and activity

Mariantonia Logozzi  1 , Clemente Capasso  2 , Rossella Di Raimo  1 , Sonia Del Prete  2 , Davide Mizzoni  1 , Mario Falchi  3 , Claudiu T Supuran  4 , Stefano Fais  1
Affiliations

Prostate cancer cells and exosomes in acidic condition show increased carbonic anhydrase IX expression and activity

Mariantonia Logozzi et al. J Enzyme Inhib Med Chem. 2019 Dec.

Abstract

Acidity and hypoxia are crucial phenotypes of tumour microenvironment both contributing to the selection of malignant cells under a micro evolutionistic pressure. During the tumour progression, nanovesicles, called exosomes and the metalloenzyme carbonic anhydrase IX (CA IX) affect the tumour growth and proliferation. Exosomes are released into the tumour microenvironment and spilt all over the body, while CA IX is a tumour-associated protein overexpressed in many different solid tumours. In the present study, to better understand the relationships between exosomes and CA IX, it has been used an in vitro cellular model of cells cultured in different pH conditions. The results showed that the acidic microenvironment induced upregulation of both expression and activity of CA IX in cancer cells and their exosomes, together with increasing the number of released exosomes. These data strongly support the importance of CA IX as a cancer biomarker and as a valuable target of new anticancer therapies.

Keywords: LNCaP; Tumour microenvironment; cancer; carbonic anhydrase IX; exosomes.

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Figures

Figure 1.
Figure 1.
Confocal microscopy analysis of CA IX expression in LNCaP cell line cultured at pH 7.4 and pH 6.5. (a)LNCaP cells cultured at pH 7.4 showed low expression of CA IX (green signal) predominately nuclear (blue signal of DAPI). (b) CA IX expression in LNCaP pH 6.5 is higher compared to pH 7.4 and showed a strong cytoplasmic expression.
Figure 2.
Figure 2.
Western Blot of lysates from LNCaP pH 7.4 and LNCaP pH 6.5 cell and exosomes. CA IX expression was analysed in LNCaP lysates from cells and exosomes cultured at different condition. CA IX expression in increased both in cells and exosomes cultured at pH 6.5 compared to samples at pH 7.4. In particular, there is an increase of CA IX 54KDa band in samples at pH 6.5. Membranes were also incubated with anti-GAPDH, a housekeeping protein, and anti-Tsg101, a typical exosomal marker.
Figure 3.
Figure 3.
Analysis of CAIX enzymatic activity. CA IX activity is shown for LNCaP cells and their exosomes (ExoLNCaP) obtained from cellular cultures grown at pH 6.5 and 7.4. The results reflect a mean of three independent experiments.
Figure 4.
Figure 4.
CA IX expression is related to microenvironmental pH. (a) Cells gradually cultured from pH 7.4 to 6.5 showed an increased CA IX expression. (b) CA IX decreased expression in LNCAP cells from pH 6.5 to 7.4. (c) Exosomes isolated from cells cultured from pH 7.4 to 6.5 showed an increased CA IX expression. (d) Exosomes isolated from cells cultured from pH 6.5 to 7.4 showed a decreased CA IX expression.
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