Abstract:Carcinogenesis cannot be explained only by genetic alterations, but also involves epigenetic processes. Modification of histones by acetylation plays a key role in epigenetic regulation of gene expression and is controlled by the balance between histone deacetylases (HDAC) and histone acetyltransferases (HAT). HDAC inhibitors induce cancer cell cycle arrest, differentiation and cell death, reduce angiogenesis and modulate immune response. Mechanisms of anticancer effects of HDAC inhibitors are not uniform; they may be different and depend on the cancer type, HDAC inhibitors, doses, etc. HDAC inhibitors seem to be promising anti-cancer drugs particularly in the combination with other anti-cancer drugs and/or radiotherapy. HDAC inhibitors vorinostat, romidepsin and belinostat have been approved for some T-cell lymphoma and panobinostat for multiple myeloma. Other HDAC inhibitors are in clinical trials for the treatment of hematological and solid malignancies. The results of such studies are promising but further larger studies are needed. Because of the reversibility of epigenetic changes during cancer development, the potency of epigenetic therapies seems to be of great importance. Here, we summarize the data on different classes of HDAC inhibitors, mechanisms of their actions and discuss novel results of preclinical and clinical studies, including the combination with other therapeutic modalities.

Free radicals are chemical particles containing one or more unpaired electrons, which may be part of the molecule. They cause the molecule to become highly reactive. The free radicals are also known to play a dual role in biological systems, as they can be either beneficial or harmful for living systems. It is clear that there are numerous mechanisms participating on the protection of a cell against free radicals. In this review, our attention is paid to metallothioneins (MTs) as small, cysteine-rich and heavy metal-binding proteins, which participate in an array of protective stress responses. The mechanism of the reaction of metallothioneins with oxidants and electrophilic compounds is discussed. Numerous reports indicate that MT protects cells from exposure to oxidants and electrophiles, which react readily with sulfhydryl groups. Moreover, MT plays a key role in regulation of zinc levels and distribution in the intracellular space. The connections between zinc, MT and cancer are highlighted.

The old herbal drug aristolochic acid (AA), derived from Aristolochia spp., has been associated with the development of a novel nephropathy, designated aristolochic acid nephropathy (AAN), and urothelial cancer in AAN patients. There is clear evidence that the major components of the plant extract AA, aristolochic acid I (AAI) and aristolochic acid II (AAII), both nitrophenanthrene carboxylic acids, are genotoxic mutagens forming DNA adducts after metabolic activation through simple reduction of the nitro group. Several mammalian enzymes have been shown to be capable of activating both AAI and AAII in vitro and in cells. The activating metabolism has been elucidated and is consistent with the formation of a cyclic nitrenium ion with delocalized charge leading to the preferential formation of purine adducts bound to the exocyclic amino groups of deoxyadenosine and deoxyguanosine. The predominant DNA adduct in vivo, 7-(deoxyadenosin-N(6)-yl)aristolactam I (dA-AAI), which is the most persistent of the adducts in target tissue, is a mutagenic lesion leading to AT-->TA transversions in vitro. This transversion mutation is found at high frequency in codon 61 of the H-ras oncogene in tumours of rodents induced by AAI, suggesting that dA-AAI might be the critical lesion in the carcinogenic process in rodents. DNA-binding studies confirmed that both AAs bind to the adenines of codon 61 in the H-ras mouse gene and preferentially to purines in the human p53 gene. In contrast, the molecular mechanism of renal interstitial fibrosis in humans after chronic administration of AA remains to be explored. However, preliminary findings suggest that DNA damage by AA is not only responsible for the tumour development but also for the destructive fibrotic process in the kidney. It is concluded that there is significant evidence that AA is a powerful nephrotoxic and carcinogenic substance with an extremely short latency period, not only in animals but also in humans. In particular, the highly similar metabolic pathway of activation and resultant DNA adducts of AA allows the extrapolation of carcinogenesis data from laboratory animals to the human situation. Therefore, all products containing botanicals known to or suspected of containing AA should be banned from the market world wide.

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Ellipticine is an antineoplastic agent, the mode of action of which is considered to be based on DNA intercalation and inhibition of topoisomerase II. We found that ellipticine also forms the cytochrome P450 (CYP)-mediated covalent DNA adducts. We now identified the ellipticine metabolites formed by human CYPs and elucidated the metabolites responsible for DNA binding. The 7-hydroxyellipticine, 9-hydroxyellipticine, 12-hydroxyellipticine, 13-hydroxyellipticine, and ellipticine N 2 -oxide are generated by hepatic microsomes from eight human donors. The role of specific CYPs in the oxidation of ellipticine and the role of the ellipticine metabolites in the formation of DNA adducts were investigated by correlating the levels of metabolites formed in each microsomal sample with CYP activities and with the levels of the ellipticine-derived deoxyguanosine adducts in DNA. On the basis of this analysis, formation of 9-hydroxyellipticine and 7-hydroxyellipticine was attributable to CYP1A1/2, whereas production of 13-hydroxyellipticine and ellipticine N 2 -oxide, the metabolites responsible for formation of two major DNA adducts, was attributable to CYP3A4. Using recombinant human enzymes, oxidation of ellipticine to 9-hydroxyellipticine and 7-hydroxyellipticine by CYP1A1/2 and to 13-hydroxyellipticine and N 2 -oxide by CYP3A4 was corroborated. Homologue modeling and docking of ellipticine to the CYP3A4 active center was used to explain the predominance of ellipticine oxidation by CYP3A4 to 13-hydroxyellipticine and N 2 -oxide.

Many studies using mammalian cellular and subcellular systems have demonstrated that polycyclic aromatic hydrocarbons, including benzo[a]pyrene (BaP), are metabolically activated by cytochrome P450s (CYPs). In order to evaluate the role of hepatic versus extra-hepatic metabolism of BaP and its pharmacokinetics, we used the hepatic cytochrome P450 reductase null (HRN) mouse model, in which cytochrome P450 oxidoreductase, the unique electron donor to CYPs, is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated intraperitoneally (i.p.) with 125 mg/kg body wt BaP daily for up to 5 days. Clearance of BaP from blood was analysed by high-performance liquid chromatography with fluorescence detection. DNA adduct levels were measured by (32)P-post-labelling analysis with structural confirmation of the formation of 10-(deoxyguanosin-N(2)-yl)-7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene by liquid chromatography-tandem mass spectrometry analysis. Hepatic microsomes isolated from BaP-treated and untreated mice were also incubated with BaP and DNA in vitro. BaP-DNA adduct formation was up to 7-fold lower with the microsomes from HRN mice than with that from WT mice. Most of the hepatic microsomal activation of BaP in vitro was attributable to CYP1A. Pharmacokinetic analysis of BaP in blood revealed no significant differences between HRN and WT mice. BaP-DNA adduct levels were higher in the livers (up to 13-fold) and elevated in several extra-hepatic tissues of HRN mice (by 1.7- to 2.6-fold) relative to WT mice. These data reveal an apparent paradox, whereby hepatic CYP enzymes appear to be more important for detoxification of BaP in vivo, despite being involved in its metabolic activation in vitro.

Balkan endemic nephropathy (BEN) is found in certain rural areas of the Balkans and affects at least 25,000 inhabitants. Of the many hypotheses on BEN, the Aristolochia hypothesis has recently gained ground substantiated by the investigations on aristolochic acid nephropathy (AAN). On both clinical and morphological grounds, AAN is very similar to BEN. That exposure to aristolochic acid (AA) of individuals living in endemic areas through consumption of bread made with flour contaminated with seeds of Aristolochia clematitis is responsible for BEN is an old hypothesis, but one which is fully consistent with the unique epidemiologic features of BEN. Here, we propose an approach to investigate AA-induced mutagenesis in BEN that can provide molecular clues to the aetiology of its associated urothelial cancer. The molecular mechanism of AA-induced carcinogenesis demonstrates a strong association between DNA adduct formation, mutation pattern and tumour development. A clear link between urothelial tumours, p53 mutations and AA exposure should emerge as more tumour DNA from BEN patients from different endemic areas becomes available for mutation analysis. We predict that the observed p53 mutation spectrum will be dominated by AT --> TA transversion mutations as has already been demonstrated in the human p53 gene of immortalized cells after exposure to AAI and urothelial tumours from BEN patients in Croatia. Moreover, the detection of AA-specific DNA adducts in renal tissue of a number of BEN patients and individuals living in areas endemic for BEN in Croatia provides new evidence that chronic exposure to AA is a risk factor for BEN and its associated cancer.

3-Nitrobenzanthrone (3-nitro-7H-benz [de]anthracen-7-one, 3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust and air pollution. We compared the ability of human hepatic cytosolic samples to catalyze DNA adduct formation by 3-NBA. Using the 32 P-postlabeling method, we found that 12/12 hepatic cytosols activated 3-NBA to form multiple DNA adducts similar to those formed in vivo in rodents. By comparing 3-NBA-DNA adduct formation in the presence of cofactors of NAD(P)H:quinone oxidoreductase (NQO1) and xanthine oxidase, most of the reductive activation of 3-NBA in human hepatic cytosols was attributed to NQO1. Inhibition of adduct formation by dicoumarol, an NQO1 inhibitor, supported this finding and was confirmed with human recombinant NQO1. When cofactors of N,Oacetyltransferases (NAT) and sulfotransferases (SULT) were added to cytosolic samples, 3-NBA-DNA adduct formation increased 10-to 35-fold. Using human recombinant NQO1 and NATs or SULTs, we found that mainly NAT2, followed by SULT1A2, NAT1, and, to a lesser extent, SULT1A1 activate 3-NBA. We also evaluated the role of hepatic NADPH:cytochrome P450 oxidoreductase (POR) in the activation of 3-NBA in vivo by treating hepatic POR-null mice and wild-type littermates i.p. with 0.2 or 2 mg/kg body weight of 3-NBA. No difference in DNA binding was found in any tissue examined (liver, lung, kidney, bladder, and colon) between null and wild-type mice, indicating that 3-NBA is predominantly activated by cytosolic nitroreductases rather than microsomal POR. Collectively, these results show the role of human hepatic NQO1 to reduce 3-NBA to species being further activated by NATs and SULTs. (Cancer Res 2005; 65(7): 2644-52)