Synthesis of sulforaphane and inhibition of cytochrome P450 enzymes as a basis for antigenotoxicity

Many dietary factors have been associated with a decreased risk of developing cancer. One potential mechanism by which these factors, chemopreventors, protect against cancer may be via alteration of carcinogen metabolism. The broccoli constituent sulforaphane (1-isothiocyanate-4-methylsulinylbutane) (CH3-S0-(CH2)4-NCS) has been isolated as a potential inducer of phase II detoxification enzymes and also protects rodents against 9,10-dimethyl-1,2-benz[aJanthracene-induced mammary tumours. The ability of sulforaphane to also modulate phase I activation enzymes (cytochrome P450) (CYP450) was studied here. Sulforaphane was synthesised with an overall yield of 15%, essentially via 1-methylsulfinylphthalimidobutane, which was oxidised to the sulfoxide moiety. Deprotective removal of phthalimide yielded the amine, which was converted into sulforaphane by reaction with N,N'-thionocarbonyldiimidazole. Purity (95 %) was checked by 1H-NMR,13C-NMR and infrared and mass spectrometry.Sulforaphane was a competitive inhibitor of CYP2E1 in acetone-induced Sprague-Dawley rat microsomes (Ki 37.9 ± 4.5μM), as measured by the p-nitrophenol hydroxylase assay. Ethoxyresorufin deethylase activity (EROD), a measurement of CYP1A activity, was also inhibited by sulforaphane (100μM) but was not competitive, and a preincubation time-dependence was observed. In view of these results, the capacity of sulforaphane to inhibit N-nitrosodimethylamine (NDMA)-induced genotoxicity (CYP2E1-mediated) was studied using mouse liver activation systems. Sulforaphane (>0.8μM) inhibited the mutagenicity of NDMA (4.4 mg/plate) in Salmonella typhimurium strain TA100 after pre-incubation for 45 min with acetone-induced liver 9000 g supernatants from Balb/c mice. Unscheduled DNA synthesis induced by NDMA (33μ5 M) in mouse hepatocytes was also reduced by sulforaphane in a concentration-dependent manner (0.064-20μM). Sulforaphane was not genotoxic itself in any of these systems and cytotoxic only at high concentrations (>0.5 mM and > 40μM respectively). The ability of sulforaphane to modulate the orthologous human enzymes was studied using a human epithelial liver cell line (THLE) expressing individual human CYP450 isoenzymes. Using the Comet assay (a measurement of DNA strand breakage under alkaline conditions), NDMA (0.01-1μg/ml) and IQ (0.1-10μg/ml) were used to produce strand breaks in T5-2E1 cells (expressing human CYP2E1) and T5-1A2 cells (expressing human CYP1A2) respectively, however no response was observed in T5-neo cells (without CYP450 cDNA transfection). Sulforaphane inhibited both NDMA and IQ-induced DNA strand breakage in a concentration-dependent manner (0.1-10μM).The inhibition of metabolic activation as a basis for the antigenotoxic action of sulforaphane in these systems (bacteria, rodent hepatocytes and human cells) is further supported by the lack of this chemopreventor to influence NaN3 mutagenicity in S. typhimurium and H202-induced DNA strand breakage in T5-neo cells. These findings suggest that inhibition of CYP2E1 and CYP1A by sulforaphane may contribute to its chemoprotective potential.

Evaluation of DNA enzymes targeted against the RNA component of human telomerase

Glioblastoma Multiforme (GBM) is a highly malignant form of brain cancer for which there is currently no effective cure. Consequently, developing new therapies and elucidating effective targets is crucial for this fatal disease. In recent years, DNA enzymes, deoxyribonucleic acid molecules with enzymatic activity, have emerged. In the same manner as ribozymes, DNA enzymes are able to effect cleavage of RNA in a sequence-specific manner, and operate with catalytic efficiency. In this study, two DNA enzymes were designed to target the template region of human telomerase RNA (hTR), utilising the 10-23 and 8-17 catalytic motifs elucidated by Santoro and Joyce (1997). Telomerase is an RNA-dependent DNA polymerase, which stabilises telomere lengths by adding hexameric repeats (TTAGGG in humans) to chromosome termini, thus preventing the telomere shortening that usually occurs during mitotic cell division. Telomerase activity, whilst absent in normal somatic tissues, is present in almost 90% of all tumours. Thus, there is speculation that telomerase may be the much sought universal target for therapeutic intervention in cancer. In vitro cleavage assays showed both DNA enzymes to be catalytically competent. Unmodified phosphodiester (PO) backbone DNA enzymes were rapidly degraded in the presence of serum, with a half-life of 10 minutes. The common approach of introducing phosphorothioate (PS) linkages was used in an effort to overcome this instability. As a result of concurrent activity and stability studies on the DNA enzymes with various numbers of PS linkages, the DNA enzymes with a PO core and PS arms were chosen for use in further cell work. The cleavage activity of both was shown to be specific and affected by temperature, pH, MgCI2 concentration and enzyme concentration. Both DNA enzyme motifs reduced telomerase activity in cell lysates, as assessed by the telomerase repeat amplification protocol (TRAP) with an IC50 of 100nM. DNA enzymes being polyanionic molecules do not readily cross biological barriers. Cellular association of naked DNA enzyme was inefficient at less than 2%. Cellular delivery of the DNA enzymes was effectively improved using commercial cationic lipid formulations. However, the lipid-mediated delivery of DNA enzymes to U87-MG cells over a 4-hour period did not significantly inhibit cell proliferation compared to controls. This is possibly due to an expected lag period between the inhibition of telomere maintenance and cell death. Therefore, biodegradable polymer microspheres were investigated as a potential delivery option for prolonged and sustained delivery. In vitro release profiles showed that after an initial burst, sustained release of DNA enzymes was observed over 35 days. Finally, the efficacy and specificity of the DNA enzymes were demonstrated in a luciferase based reporter assay. Specific inhibition of luciferase expression was displayed at 10nM. Thus DNA enzymes have potential against endogenous cellular targets.

NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure

Markers of increased oxidative stress are known to be elevated following acute myocardial infarction and in the context of chronic left ventricular hypertrophy or heart failure, and their levels may correlate with the degree of contractile dysfunction or cardiac deficit. An obvious pathological mechanism that may account for this correlation is the potential deleterious effects of increased oxidative stress through the induction of cellular dysfunction, energetic deficit or cell death. However, reactive oxygen species have several much more subtle effects in the remodelling or failing heart that involve specific redox-regulated modulation of signalling pathways and gene expression. Such redox-sensitive regulation appears to play important roles in the development of several components of the phenotype of the failing heart, for example cardiomyocyte hypertrophy, interstitial fibrosis and chamber remodelling. In this article, we review the evidence supporting the involvement of reactive oxygen species and redox signalling pathways in the development of cardiac hypertrophy and heart failure, with a particular focus on the NADPH oxidase family of superoxide-generating enzymes which appear to be especially important in redox signalling.