Carbonic anhydrase inhibitor suppresses invasion of renal cancer cells in vitro

Abstract

Acidification of the extracellular milieu of malignant tumors is reported to increase the invasive behavior of cancer cells. In normal tissues, production of acid is catalyzed by carbonic anhydrases (CAs), some of which are known to be overexpressed in certain cancers. To investigate the functional role of CA activity in such cancer cells, we analyzed the effect of acetazolamide, a potent CA inhibitor, on the invasive capacity of four renal carcinoma cell lines (Caki-1, Caki-2, ACHN, and A-498). We found that 10 microM acetazolamide inhibited the relative invasion rate of these cell lines between 18-74%. The Caki-2 and ACHN cell lines displayed the highest responsiveness, and their responses clearly depended on the acetazolamide concentration in the culture medium. Immunocytochemical and Western blotting results identified the presence of CA isoenzyme II in the cytoplasm of all four cell lines and CA XII on the plasma membrane in three of four cell lines. Because acetazolamide alone reduced invasiveness of these cancer cells in vitro, we conclude that the CAs overexpressed in these renal cancer cells contribute to invasiveness, at least in vitro, and suggest that CA inhibitors may also reduce invasiveness in other tumors that overexpress one or more CAs.

Figure 1

Effect of 10 μM acetazolamide on cell invasion rate. The invasion assay was repeated three (Caki-1 and A-498 cells) or four (Caki-2 and ACHN cells) times. The results are shown as means ± SEM.

Figure 2

Effect of acetazolamide concentration on the invasion rate in Caki-2 and ACHN cells.

Figure 3

Effect of 100 μM acetazolamide on cell proliferation in ACHN cells. The results are shown as means ± SEM.

Figure 4

Immunocytochemical staining of renal cancer cells for CA II, IV, IX, and XII. Caki-1, Caki-2, and A-498 cells show strong positive staining for CA II, whereas the signal in ACHN cells is less intense. None of the cell lines express CA IV. Only A-498 cells show positive immunostaining for CA IX, whereas Caki-2, ACHN, and A-498 are all positive for CA XII.

Figure 5

Western blotting of Caki-1, Caki-2, ACHN, and A-498 cells with antibodies raised against human CA II, IV, IX, and XII. The results confirm the pattern of expression of different CA isoenzymes in renal cancer cells as indicated by immunocytochemistry (compare with Fig. 4). MM, molecular mass.