Further evidence of the importance of the CAs in the oncogenic process comes from the use of CA inhibitors, most notably the heterocyclic and aromatic sulphonamides of which acetazolamide is the most prominent.
In bicarbonate-free medium, sulphonamides were effective in reducing growth rates of lymphoma cell lines according to their order of potency in CA inhibition (Chegwidden et al, 2000
). The growth inhibition was reversed by supplementing the medium with nucleotide precursors, suggesting that these may be depleted most readily by the high metabolic flux of the transformed cell in a low bicarbonate environment. The critical step may involve carbamoyl synthetase II, a cytosolic enzyme that utilises bicarbonate for the first step in pyrimidine nucleotide biosynthesis.
Many novel sulphonamide derivatives have been shown to have growth-inhibitory effects on a variety of malignant cell lines in vitro
, often at concentrations in the nanomolar range (Supuran et al, 2001
). However, some sulphonamides without any CA-inhibitory activity also show growth-inhibitory effects, possibly because of inhibition of tubulin polymerisation. There is also potentially a degree of crossover with inhibition of other zinc metalloenzymes, notably the matrix metalloproteinases.
Acetazolamide has been shown to reduce invasiveness of four RCC cell lines in vitro
by 18–74%, although the only cell line shown to express CAIX was also the least affected (Parkkila et al, 2000b
). The concentrations used were orders of magnitude greater than that needed to inhibit CA.
Invasiveness is known to increase in cells cultured in low pH medium, and it is possible that the presence of CA activity may promote this acidification of the extracellular space, with concomitant activation of enzymes required for matrix degradation (Martinez-Zaguilan et al, 1996
Acetazolamide has shown antitumour properties in a murine fibrosarcoma model, producing significant growth delays when used as a single agent, and additive growth delays in combination with a number of chemotherapeutic agents (Teicher et al, 1993
In the clinical setting, the differential expression of CAIX in renal cancers has provided a target for radioimmunotherapy, antibody-mediated gene transfer and vaccination strategies (Tso et al, 2001
), and phase I/II trial results with iodine-131 labelled murine monoclonal antibodies have been performed with some antitumour activity (Divgi et al, 1998
). The development of a humanised monoclonal antibody is awaited to allow repeated cycles of treatment.
Many chemotherapeutic drugs are weak acids or bases, their pKa
's being in the physiological range. As most of these drugs enter the cell by passive diffusion and the plasma membrane is relatively impermeable to ionised species, slight differences in pH on either side of the membrane may result in dramatic alterations in the distribution of the drug due to ion trapping. In areas of tumour distant from the vasculature, extracellular pH may be expected to drop, whereas the intracellular pH remains relatively constant, increasing the intra- to extracellular pH gradient and favouring the uptake of weak acid drugs. Many clinically useful chemotherapeutic drugs are weak bases (e.g. doxorubicin, bleomycin, mitoxantrone), whose uptake in animal models may be enhanced by bicarbonate administration, reducing the extracellular acidity of tumours (Raghunand and Gillies, 2001
). Whether CA inhibitors may be effective in reducing this tumour acidity and consequently have a role in combination chemotherapy remains to be demonstrated.