One of the most prominent alterations in cancer cells is their strict dependence on the glycolytic pathway for ATP generation. This observation led to the evaluation of glycolysis inhibitors as potential anticancer agents. The inhibition of lactate dehydrogenase (LDH) is a promising way to inhibit tumor cell glucose metabolism without affecting the energetic balance of normal tissues. However, the success of this approach depends chiefly on the availability of inhibitors that display good selectivity. We identified a compound (galloflavin, CAS 568-80-9) which, in contrast to other inhibitors of human LDH, hinders both the A and B isoforms of the enzyme. To determine the mechanism of action, we collected LDH-A and -B inhibition data in competition reactions with pyruvate or NADH and evaluated the results using software for enzyme kinetics analysis. We found that galloflavin inhibits both human LDH isoforms by preferentially binding the free enzyme, without competing with the substrate or cofactor. The calculated Ki values for pyruvate were 5.46 μM (LDH-A) and 15.06 μM (LDH-B). In cultured tumor cells, galloflavin blocked aerobic glycolysis at micromolar concentrations, did not interfere with cell respiration, and induced cell death by triggering apoptosis. To our knowledge, the inhibition of LDH is, to date, the only biochemical effect described for galloflavin. Because galloflavin is not commercially available, we also describe herein a procedure for its synthesis and report its first full chemical characterization.

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In the attempt of developing innovative anticancer treatments, growing interest has recently focused on the peculiar metabolic properties of cancer cells. In this context, LDH, which converts pyruvate to lactate at the end of glycolysis, is emerging as one of the most interesting molecular targets for the development of new inhibitors. In fact, because LDH activity is not needed for pyruvate metabolism through the TCA cycle, inhibitors of this enzyme should spare glucose metabolism of normal non-proliferating cells, which usually completely degrade the glucose molecule to CO2. This review is aimed at summarizing the available data on LDH biology in normal and neoplastic cells, which support the anticancer therapeutic approach based on LDH inhibition. These data encouraged pharmaceutical industries and academic institutions in the search of small-molecule inhibitors and promising candidates have recently been identified. The availability of inhibitors with drug-like properties will allow the evaluation in the near future of the real potential of LDH inhibition in anticancer treatment, also making the identification of the most responsive neoplastic conditions possible.

The asialoglycoprotein receptor (ASGP-R) is a glycoprotein present in large amount only on hepatocytes where it is expressed on the sinusoidal and lateral plasma membrane (Morell et al, 1968;Geffen and Spiess, 1992). ASGP-R binds and internalizes a broad range of molecules exposing galactose or N-acetyl-galactosamine residues. Following internalization the fate of ligand is lysosomal degradation (Morell et al, 1968;Geffen and Spiess, 1992).Taking advantage of this receptor a chemotherapeutic approach has been developed to reduce the extrahepatic side-effects of nucleoside analogues (NAs) used in chronic viral hepatitis (Fiume et al, 1979(Fiume et al, , 1997 Torriani et al, 1996). These drugs are coupled to galactosyl-terminating peptides. The conjugates selectively enter hepatocytes, where the lysosomal enzymes split the bond between the carrier and the drug, which becomes concentrated in liver cells. A similar strategy was suggested in order to increase the chemotherapeutic index of drugs inhibiting DNA synthesis in the treatment of human hepatocellular carcinoma (HCC) (Schneider et al, 1984;O'Hare et al, 1989;Di Stefano et al, 1998). This approach obviously requires the presence of the receptor in the neoplastic tissue and maintenance of its expression on DNA synthesizing cells.Few and conflicting data are available on the distribution of ASGP-R in human neoplastic hepatocytes. In a study in which the receptor was measured by a biochemical procedure, it was not found in HCC samples (Sawamura et al, 1984), whereas in an immunohistochemical investigation carried out on ten cases of HCC the presence of the receptor was demonstrated in the more differentiated forms (Hyodo et al, 1993). In the present study we have first assessed the frequency of ASGP-R expression in a large number of human HCCs. For this purpose, needle biopsy samples of sixty consecutive HCCs were analysed. Visualization of the receptor was obtained using an anti-ASGP-R monoclonal antibody applied after antigen retrieval procedure to routinely formalinfixed, paraffin-embedded liver samples. Since we found that the ASGP-R was present in 33 HCCs we also investigated whether the ASGP-R was maintained on the plasma membrane of DNA synthesizing cancer cells. For this purpose we incubated HCC tissue samples with bromodeoxyuridine (BrdU). The presence of ASGP-R on DNA-synthesizing cancer cells was immunohistochemically assessed using anti-BrdU and anti-ASGP-R antibodies on the same tissue section. Summary The expression of the asialoglycoprotein receptor (ASGP-R) on human hepatocellular carcinoma (HCC) cells might be exploited to reduce the extrahepatic toxicity of DNA synthesis inhibitors by their conjugation with galactosyl-terminating peptides. In the present study we first assessed the frequency of ASGP-R expression in 60 HCCs. Secondly, we investigated whether the receptor was maintained on the plasma membranes of DNA synthesizing cancer cells. Needle biopsies of HCC were evaluated. Diagnosis and grading of HCC were performed on routine haematoxyli...

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1. Injection of alpha-amanitin to mice causes a decreased incorporation of [6-(14)C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn(2+) and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with alpha-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg(2+)-activated RNA polymerase is only slightly affected by alpha-amanitin either administered to mice or added in vitro.

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