Tumor-associated Isozymes Research Articles

Carbonic anhydrase inhibitors. Inhibition of tumor-associated isozyme IX by halogenosulfanilamide and halogenophenylaminobenzolamide derivatives.

Two series of halogenated sulfonamides have been prepared. The first consists of mono/dihalogenated sulfanilamides, whereas the second one consists of the mono/dihalogenated aminobenzolamides, incorporating equal or different halogens (F, Cl, Br, and I). These sulfonamides have been synthesized from the corresponding anilines by acetylation (protection of the amino group), chlorosulfonylation, followed either by amidation, or reaction with 5-amino-1,3,4-thiadiazole-2-sulfonamide (and eventually deacetylation). All these compounds, together with the six clinically used sulfonamide inhibitors (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and brinzolamide) were investigated as inhibitors of the transmembrane, tumor-associated isozyme carbonic anhydrase (CA) IX. Inhibition data against the classical, physiologically relevant isozymes I, II, and IV were also obtained. CA IX shows an inhibition profile which is generally completely different from those of isozymes I, II, and IV, with potent inhibitors (inhibition constants in the range of 12-40 nM) among both simple aromatic (such as 3-fluoro-5-chloro-4-aminobenzenesulfonamide) as well as heterocyclic compounds (such as acetazolamide, methazolamide, 5-amino-1,3,4-thiadiazole-2-sulfonamide, aminobenzolamide, and dihalogenated aminobenzolamides). This first detailed CA IX inhibition study revealed many interesting leads, suggesting the possibility to design even more potent and eventually CA IX-selective inhibitors, with putative applications as antitumor agents.

Carbonic anhydrase inhibitors: inhibition of the tumor-associated isozyme IX with aromatic and heterocyclic sulfonamides

The inhibition of the tumor-associated transmembrane carbonic anhydrase IX (CA IX) isozyme has been investigated with a series of aromatic and heterocyclic sulfonamides, including the six clinically used derivatives acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide and brinzolamide. Inhibition data for the physiologically relevant isozymes I and II (cytosolic forms) and IV (membrane-bound) were also provided for comparison. A very interesting and unusual inhibition profile against CA IX with these sulfonamides has been observed. Several nanomolar ( K I-s in the range of 14–50 nM) CA IX inhibitors have been detected, both among the aromatic (such as orthanilamide, homosulfonilamide, 4-carboxy-benzenesulfonamide, 1-naphthalenesulfonamide and 1,3-benzenedisulfonamide derivatives) as well as the heterocylic (such as 1,3,4-thiadizole-2-sulfonamide, etc.) sulfonamides examined. Because CA IX is a highly active isozyme predominantly expressed in tumor tissues with poor prognosis of disease progression, this finding is very promising for the potential design of CA IX-specific inhibitors with applications as anti-tumor agents.

Localisation of a tumor-associated phenotype of benz-aldehyde dehydrogenase in liver carcinogenesis of flounder by quantitative histochemistry

Aldehyde dehydrogenases (ALDHs) oxidize endogeneous reactive aldehydes including lipid peroxidation products (propionaldehyde, malondialdehyde) and are also inducible by xenobiotics like DDT and TCDD. ALDHs are found in injured livers and cancers of contaminant-exposed flounder (Platichthys flesus L.) and showed identical characteristics as in hepatocellular carcinomas of rats by its cytosolic localisation, the preferential oxidation of aromatic compounds (benzaldehyde), dependency on NADP as coenzyme and sensitivity to the specific inhibitor disulfiram. Image analysis showed increased activity of NADP-dependent benzaldehyde dehydrogenase localised in a few cells of early eosinophilic foci and in basophilic adenomas, carcinomas and their invasively growing protrusions (satellites). Disulfiram sensitivity to differentiate between the xenobiotic-inducible isozyme and the tumor-specific isozyme in rats was highest in the early stages of carcinogenesis in the liver of fish. Moderate disulfiram inhibition was observed in non-neoplastic and extrafocal tissue. In neoplastic livers of flounder from contaminated areas (Elbe estuary), hepatocytes in early stages of carcinogenesis were best protected against accumulation of toxic aldehydes by increased activity of the specific tumor-associated isozyme.

Aldehyde dehydrogenase and glucose-6-phosphate dehydrogenase as markers for enzyme-altered foci in the liver of dab ( Limanda limanda L.)

The activities of the most important aldehyde oxidizing enzymes, the aldehyde dehydrogenases (ALDH), were detected histochemically in liver cryosections of dab ( Limanda limanda L.) caught at differently polluted stations along the North Sea coast. In parallel, glucose-6-phospate dehydrogenase (G6PDH) as an NADPH-generating enzyme was measured. Increased activities of both enzymes localized putatively preneoplastic hepatocytes (foci), but ALDH in combination with propionaldehyde and NAD marked 50% more foci than G6PDH alone. Nevertheless, at the more contaminated stations with highest prevalences of foci, ALDH activities showed a shift in substrate and coenzyme dependence from propionaldehyde and NAD to benzaldehyde and NADP in altered hepatocytes, a possible result of physiological adaption to the toxic environment. Increased G6PDH activities in focal hepatocytes indicated elevated NADPH production in contrast to the injured extrafocal liver parenchyma showing reduced G6PDH activities compared to normal livers. Using the ALDH inhibitor disulfiram, the existence of a specific tumor-associated isozyme previously found in rats could not be clearly demonstrated in early enzyme-altered hepatocellular foci of dab. However, based on the total inhibition of Bz-NADP-dependent ALDH, the presence of a xenobiotic-induced ALDH isozyme can be anticipated.

Hepatocyte expression of tumor associated aldehyde dehydrogenase (ALDH-3) and p21 Ras following diethylnitrosamine (DEN) initiation and chronic exposure to di(2-ethylhexyl)phthalate (DHEP).

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP) either promote or inhibit rat liver tumorigenesis depending on the carcinogenesis protocol. In this study, we examined the expression of two histochemical markers, the tumor associated isozyme of aldehyde dehydrogenase (ALDH-3) and the oncoprotein p21 Ras, in the livers of male F344 rats. The rats were initiated with DEN and further treated with either DEHP (a known inhibitor of hepatocarcinogenesis), phenobarbital (PB, a known promoter of hepatocarcinogenesis), or a combination of DEHP and PB. The studies were designed to examine the expression of these markers in both normal appearing liver and hepatic hyperplastic and neoplastic lesions and to correlate the early expression of the markers at 26 weeks in the normal appearing liver to later tumor incidence at 52 weeks. The expression of each marker was detected by immunohistochemical methods on formalin-fixed paraffin embedded sections of normal appearing liver or liver lesions. We found that ALDH-3 and p21 expression were significantly enhanced in rats receiving PB after DEN initiation at 26 weeks and that the incidence of hepatocellular carcinomas was likewise increased compared to control or DEN only treated animals. DEN initiation followed by a combination of PB and either 0.1 or 0.5% DEHP significantly reduced ALDH-3 but not p21 Ras expression at 26 weeks compared to DEN plus PB only. These treatment regimens also reduced the incidence of hepatocellular carcinomas at 52 weeks. DEN followed by any of the three doses of DEHP without PB resulted in ALDH-3 expression similar to DEN alone. However, p21 Ras expression was significantly increased after these treatments. For all treatment groups, both the early (26 weeks) expression of p21 Ras and ALDH-3 correlated with hepatocellular carcinoma incidence at 52 weeks. However, the correlation between hepatocellular carcinoma and ALDH-3 expression was better than p21 Ras or the other markers we have studied. We concluded that ALDH-3 expression is significantly downregulated after DEHP treatment, and that expression of the isozyme correlated with later hepatocarcinoma incidence and may indicate a significant relationship between ALDH-3 expression and hepatocarcinogenesis during DEHP treatment.

Cytochemical detection of a class 3 aldehyde dehydrogenase in human hepatocellular carcinoma.

The development of hepatocellular carcinoma in rodents treated with different chemical compounds is associated with the appearance in the cytosol of neoplastic liver cells of an unusual aldehyde dehydrogenase isozyme of class 3 (ALDH-3) which is very active with aromatic aldehydes. This tumor-associated isozyme is readily detected by enzyme cytochemistry using the substrate benzaldehyde with NADP as coenzyme. To determine whether human hepatocellular carcinomas express ALDH-3, the activity of this isozyme was examined in frozen sections from 68 echo-guided human liver biopsies. In 54 cases the guided biopsy was performed on one or more nodules suggestive for hepatocellular carcinoma found at ultrasonography within the liver parenchyma. The remaining 14 patients were affected by chronic active hepatitis or cirrhosis. An intense enzymatic activity was ascertained in 5 out of 36 hepatocellular carcinomas. In non-neoplastic liver, in macroregenerative nodules and in metastatic adenocarcinomas enzymatic activity was not detectable. ALDH-3-positive tumors were typical hepatocellular carcinomas (histological grade II and III). These results suggest that ALDH-3 is a phenotype associated with malignancy in human liver tumors.

Carbonic anhydrase: chemistry, physiology, and inhibition.

Carbonic anhydrase: chemistry, physiology, and inhibition.