DNA methyltransferase inhibitor assay system based on the HBx-induced DNA methylation of E-cadherin

We here report a simple assay system for DNA methyltransferase (DNMT) inhibitors based on the HBx-induced DNA methylation of E-cadherin. A stable cell line named G1 was generated by co-transfecting E-cadherin luciferase reporter and HBx-expression plasmid into HepG2 cells. Treatment of G1 cells with DNMT inhibitors, 5-azacytidine, 5-aza-2′-deoxycytidine, and procainamaid, dose-dependently inhibited DNA methylation of E-cadherin promoter in the reporter, resulting in up-regulation of luciferase levels and its enzyme activity. Treatment with all-trans retinoic acid that is known to inhibit DNMT expression, also induced similar effects. Our system can be useful for development of epi-drugs targeting DNA methylation in malignancies.     

Discovery of novel DNA methyltransferase 3A inhibitors via structure-based virtual screening and biological assays

DNA methyltransferases are involved in diverse biological processes and abnormal methylation patterns play essential roles in cancer initiation and progression. DNA methyltransferase 3A (DNMT3A) acting as a de novo DNA methyltransferase, has gained widespread attention especially in haematological diseases. To date, large numbers of DNMTs inhibitors have been discovered, however, the small molecular inhibitors targeting DNMT3A are still in its infancy. In this study, structure-based virtual screening in combination with biological assays was performed to discovery potent novel DNMT3A inhibitors. Compound 40 and 40_3 displayed comparable in vitro inhibitory activity against DNMT3A with IC₅₀ values of 46.5 μM and 41 μM, respectively. Further binding mode analysis suggested these molecules inhibit DNMT3A activity through binding the S-adenosyl-l-methionine (SAM) pocket. Overall, 40 and 40_3 may serve as novel scaffolds for further optimization and small molecular probes for investigating DNMT3A function.     

Design,synthesis, inhibitory activity,and binding mode study of novel DNA methyltransferase 1 inhibitors

To identify novel non-nucleoside DNA methyltransferase (DNMT) inhibitors, we designed and synthesized a series of maleimide derivatives. Among this series, compounds 5–8 were found to be more potent DNMT1 inhibitors than RG108, a DNMT1 inhibitor reported previously by Siedlecki et al. The binding mode analysis of compound 5 is also reported.     

Metal specificity in DNA damage prevention by sulfur antioxidants

Metals such as Cu^I and Fe^II generate hydroxyl radical (OH) by reducing endogenous hydrogen peroxide (H₂O₂). Because antioxidants can ameliorate metal-mediated oxidative damage, we have quantified the ability of glutathione, a primary intracellular antioxidant, and other biological sulfur-containing compounds to inhibit metal-mediated DNA damage caused hydroxyl radical. In the Cu^I/H₂O₂ system, six sulfur compounds, including both reduced and oxidized glutathione, inhibited DNA damage with IC₅₀ values ranging from 3.4 to 12.4 μM. Glutathione and 3-carboxypropyl disulfide also demonstrated significant antioxidant activity with Fe^II and H₂O₂. Additional gel electrophoresis and UV-vis spectroscopy studies confirm that antioxidant activity for sulfur compounds in the Cu^I system is attributed to metal coordination, a previously unexplored mechanism. The antioxidant mechanism for sulfur compounds in the Fe^II system, however, is unlike that of Cu^I. Our results demonstrate that glutathione and other sulfur compounds are potent antioxidants capable of preventing metal-mediated oxidative DNA damage at well below their biological concentrations. This novel metal-binding antioxidant mechanism may play a significant role in the antioxidant behavior of these sulfur compounds and help refine understanding of glutathione function in vivo.     

Strand scission in DNA by Gossypol and Cu(II): Role of Cu(I) and oxygen-free radicals

Gossypol, a polyphenolic binaphthyl dialdehyde found in cotton seeds, is a dietary mutagen and a potential male contraceptive. In the presence of Cu(II), gossypol caused breakage of supercoiled plasmid pBR322 DNA. The products were relaxed circles or a mixture of these and linear molecules. Other metal ions tested [Ni(II), Co(II), Mn(II), and Fe(II)] were ineffective or less effective in the DNA breakage reaction. In the case of gossypol-Cu(II) mediated cleavage, (Cu(I) was shown to be an essential intermediate by using the Cud) sequestering reagent bathocuproine. By using job plots, it was established that in the absence of DNA, eight Cu(II) ions can be reduced by one gossypol molecule. The involvement of active oxygen species, such as singlet oxygen and H₂O₂, was established by the inhibition of DNA breakage by catalase and by sodium azide. It was further shown that gossypol is capable of directly producing H₂O₂.     

Studies on the structure—activity relationship among aliphatic and aromatic bisguanylhydrazones and some related compounds

A total of 18 compounds consisting of 7 aliphatic and 7 aromatic bis(guanylhydrazones), p-quinone-bis(guanylhydrazone), one monoguanylhydrazone, one diamidine and one diguanidine were studied spectrophotometrically to determine their ability to interact with native calf-thymus DNA and the possible correlation of binding with biological activity. In each case, the ability of a compound to bind to DNA correlated with its ability to inhibit the activity of DNA-dependent DNA polymerase (EC 2.7.7.7) extracted from mouse leukemia L1210 cells. For example, all the aromatic bis-guanylhydrazones and diamidine (hydroxystilbamidine), which were good inhibitors of the enzyme activity, showed a biphasic interaction with DNA. All the aliphatic compounds displayed no detectable interaction with DNA in the Tris buffer used, and were also poor inhibitors of the polymerase activity. Interaction of decamethylene diguanide (Synthalin) with DNA could not be determined because the compound does not absorb light in the UV-VIS region. However, in similarity with other aliphatic compounds, this agent was a poor inhibitor of DNA polymerase reaction. The p-quinone-bis(guanylhydrazone) and p-phenylbenzaldehyde-monoguanylhydrazone showed only a monophasic interaction with DNA and caused an intermediate inhibition of the enzyme activity. When tested for possible anti-leukemic activity against i.p. L1210 leukemia in syngeneic DBA/2J mice, all the aromatic bisguanylhydrazones as well as hydroxystilbamidine caused prolongation of survival of tumor-bearing mice. Among the aliphatic bisguanylhydrazones, all of which showed no binding to DNA and caused at the most only a very slight inhibition of DNA polymerase, only methylglyoxal-bis(guanylhydrazone) (CH₃-G) had antileukemic activity. Synthalin also inhibited leukemic growth. Evidences presented indicate that the mechanisms of action of aliphatic and aromatic bisguanylhydrazones may be quite different. Furthermore, the ability to bind to DNA may be a useful criterion to predict the antileukemic activity of aromatic guanylhydrazones and possibly other aromatic bis-cationic compounds, but not that of aliphatic congeners.     

Reaction of sperm with egg-derived hydrogen peroxide helps prevent polyspermy during fertilization in the sea urchin

Recent evidence suggests roles for egg derived hydrogen peroxide (H₂O₂) and ovoperoxidase (secreted by cortical granules) in both fertilization envelope hardening and the block to polyspermy in sea urchins. Strongylocentrotus purpuratus eggs were found to release H₂O₂ during the cortical reaction at fertilization. Treatment of sperm with equivalent concentrations of H₂O₂ resulted in a rapid loss of sperm fertilizing ability. Attempts were made to induce polyspermy by utilizing ovoperoxidase inhibitors at concentrations known to inhibit fertilization envelope hardening. Eggs fertilized in phenylhydrazine became polyspermic, while 3-amino-1,2,4-triazole-treated eggs did not. These data suggested that a sperm peroxidase might be involved in preventing polyspermy. This hypothesis was tested by the addition of phenylhydrazine or 3-amino-1,2,4-trizaole to H₂O₂-treated sperm. Phenylhydrazine acted to protect sperm fertility from H₂O₂, while 3-amino-1,2,4-triazole increased the adverse effect of H₂O₂. Simultaneous addition of both inhibitors to sperm incubated in H₂O₂ gave an intermediate value of sperm fertility. These data indicate that (1) H₂O₂ generated by sea urchin eggs during the cortical reaction at fertilization is used for two separate processes, fertilization envelope hardening and the prevention of polyspermy; (2) ovoperoxidase is probably not involved in preventing polyspermy; and (3) egg-derived H₂O₂ reacts directly with sperm enzymes to prevent polyspermy. The phenylhydrazine-sensitive enzyme in the sperm is probably a peroxidase that acts to inactivate sperm, while the 3-amino-1,2,4-triazolesensitive enzyme is probably a catalase which protects sperm from H₂O₂. This hypothesis is consistent with model experiments on horseradish peroxidase and bovine liver catalase.     

Rapid breakdown of exogenous extracellular hydrogen peroxide by lichens

All organisms, even highly stress‐tolerant lichens, produce a variety of reactive oxygen species (ROS) during and after stress. Furthermore, the cell walls of some lichens in Suborder Peltigerineae contain laccases, and therefore can produce quinone radicals that can break down to yield ROS. While the extracellular ROS produced by these enzymes probably play important roles in the biology of these lichens, they may also be potentially harmful and need to be rapidly broken down. To test this, rates of breakdown of exogenously supplied H₂O₂ were measured in a range of lichen species. Considerable diversity existed in rates of H₂O₂ breakdown but rates were on average almost double in members of Suborder Peltigerineae. While all lichens tested appeared to lack extracellular peroxidases and catalases, enzymes normally involved in breaking down H₂O₂, extracellular tyrosinase activity could be readily detected in the Peltigerineae. A role for tyrosinases in H₂O₂ breakdown was supported by the results from experiments involving inhibitors, and demonstration of the simultaneous release into an incubation solution of tyrosinase activity and the ability to breakdown H₂O₂. Rates of breakdown were very high, and tyrosinase appeared to break down H₂O₂ by a catalase‐like mechanism. However, significant rates of breakdown of H₂O₂ also occurred in species that did not possess cell wall redox enzymes. These species probably took up the exogenously supplied H₂O₂ intracellularly and then broke it down by the usual catalases and peroxidases. The importance of H₂O₂ degradation is discussed in terms of its possible role in defence against the harmful effects of ROS.     

Protective effect of vanillic acid in hydrogen peroxide-induced oxidative stress in D.Mel-2 cell line

The overproduction of reactive oxygen species (ROS) causes oxidative stress, such as Hydrogen peroxide (H₂O₂). Acute oxidative stress is one of the main reasons for cell death. In this study, the antioxidant properties of vanillic acid- a polyphenolic compound was evaluated. Therefore, this study aims to check the effectiveness of vanillic acid in H₂O₂-induced oxidative stress in D. Mel-2 cell line. The efficacy was determined by biochemical tests to check the ROS production. The cytotoxicity of H₂O₂ and vanillic acid was checked by MTT assay. The DNA fragmentation was visualized by gel electrophoresis. Protein biomarkers of oxidative stress were analyzed by western blotting. The results depict a promising antioxidant effect of vanillic acid. The IC₅₀ value of vanillic acid and H₂O₂ was found 250 μg/ml and 125 μg/ml, respectively. The catalase activity, SOF, GPx, and PC was seen less in H₂O₂ treated group compared with the control and vanillic acid treated group. However, the TBRAS activity was hight in H₂O₂ treated group. The effect of H₂O₂ on DNA fragmentation was high as compared with vanillic acid-treated cells. The protein expression of Hsp70, IL-6 and iNOS was seen significant in a vanillic acid-treated group as compared with H₂O₂ treated group. These results reinforce that at low concentration, vanillic acid could be used as an antioxidant agent in the food and pharmaceutical industries.     

Inhibition of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway decreases DNA methylation in colon cancer cells

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) pathway is a critical intermediary for cell proliferation, differentiation, and survival. In the human colon cancer cell line SW1116, treatment with the DNA methyltransferase 1 (DNMT1) inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) or the ERK-MAPK inhibitors PD98059 or rottlerin, or transient transfection with the MAP/ERK kinase (MEK)1/2 small interfering RNA down-regulates DNMT1 and proliferating cell nuclear antigen levels. In this report, we found that drug treatment or small interfering RNA transfection of SW1116 cells induced promoter demethylation of the p16(INK4A) and p21(WAF1) genes, which up-regulated their mRNA and protein expression levels. Flow cytometry revealed that rottlerin treatment induced cell cycle arrest at phase G(1) (p < 0.05). Thus, the ERK-MAPK inhibitor treatment or siRNA-mediated knockdown of ERK-MAPK decreases DNA methylation via down-regulating DNMT1 expression and other unknown mediator(s) in SW1116 colon cancer cells.     

Quantification of hydrogen peroxide in plant extracts by the chemiluminescence reaction with luminol

The chemiluminescence of luminol (3-aminophthalhydrazide) with H₂O₂ has been used to quantify endogenous amounts of H₂O₂ in plant tissues. The reaction is linear over at least three orders of magnitude between 10^?5 and 10^?2M H₂O₂. Interference by coloured compounds in the crude extract is calibrated by a purification step with Dowex AG 1-X8. The extract is calibrated with an internal H₂O₂ standard, and the specificity verified by H₂O₂ purging with catalase. The minimum delectability for H₂O₂ of this assay is at least 1 ng, corresponding to 0.1–1 g fresh material. Data are presented for the levels of H₂O₂ in potatoes after treatment with oxygen and ethylene, in tomatoes before and after ripening and in untreated germinating castor beans as well as in beans treated with aminotriazol to inhibit catalase activity. Though data using the titanium test are generally confirmed, the method presented here has the advantage of higher sensitivity and specificity.     

Purification and some properties of human DNA-O6-methylguanine methyltransferase

DNA-O⁶-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1–2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O⁶-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8.5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent andE. coli methyltransferases, needs no cofactor. While lmM MnCl₂, lmM spermidine, 5mM MgCl₂ and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1Cl₃ or FeCl₂ or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with single-stranded DNA being more inhibitory than duplex DNA.     

Electrochemical detection of natural DNA damage induced by ferritin/ascobic acid/H2O2 system and amplification of DNA damage by endonuclease Fpg

Oppositely charged natural DNA and chitosan (CS) were assembled into (CS/DNA)_n layer-by-layer films on electrode surface, and Ru(bpy)₃²⁺ (bpy = bipyridyl) in solution was used as electroactive catalyst to detect damage of DNA in the films after incubation of the films in ferritin/AA/H₂O₂ solutions (AA = ascorbic acid). The mechanism of DNA damage caused by the ferritin/AA/H₂O₂ system was similar to that of Fenton reaction, where the reaction of ferritin with AA would release some Fe(II) ions from ferritin and the following reaction between Fe(II) ions and H₂O₂ would produce hydroxyl radical, which could induce DNA oxidative damage. This system provided an in vitro model to imitate the DNA damage indirectly induced by ferritin in real bio-systems. In addition, formamidopyrimidine DNA glycosylase (Fpg), a key endonuclease enzyme in repair of oxidatively damaged DNA, was used to amplify the DNA damage caused by ferritin/AA/H₂O₂ system through conversion of oxidative purine bases into single-strand breaks. The high sensitivity of electrocatalytic method with Ru(bpy)₃²⁺ as the catalyst in detection of DNA damage and the magnification function of Fpg may provide a novel idea to detect natural DNA lesion sensitively.     

Identification of a soluble protein stimulator of plasmalogen biosynthesis and stearoyl-coenzyme a desaturase

Stimulation of the desaturation of 1-alkyl-2-acyl-sn-glycero-3-phosphoethanolamine (GPE), which forms ethanolamine plasmalogens, by a component of the 105,000g supernatant has been previously reported. We have isolated the stimulatory protein and identified it as catalase. Purified rat liver catalase or commercial bovine liver catalase is as effective in stimulating microsomal 1-alkyl-2-acyl-GPE desaturation as the soluble proteins. The stimulatory effect of these proteins is eliminated by catalase inhibitors. It appears that catalase stimulates the desaturation of 1-alkyl-2-acyl-GPE by preventing inactivation of the enzyme system by H₂O₂ or a decomposition product of H₂O₂. The cytochrome b₅ content and NADH oxidation are depressed in Fischer R-3259 sarcoma microsomes by H₂O₂; this effect is eliminated by catalase. However, since measurable inhibition of 1-alkyl-2-acyl-GPE desaturase by H₂O₂ still occurred in the presence of catalase, the inhibition by H₂O₂ cannot be explained solely on the basis of cytochrome b₅ inactivation. The desaturation of stearoyl-coenzyme A, a reaction analogous in many respects to 1-alkyl-2-acyl-GPE desaturation, was also found to be stimulated by catalase.     

Mechanism of DNA Damage and Apoptosis Induced by Tetrahydropapaveroline, a Metabolite of Dopamine

Tetrahydropapaveroline (THP), a metabolite of dopamine, has been suspected to be associated with dopaminergic neurotoxicity of L-DOPA. THP induced apoptosis in human leukemia cell line HL-60 cells, but did not in its hydrogen peroxide (H₂O₂)-resistant clone HP100. THP-induced DNA ladder formation in HL-60 cells was inhibited by a metal chelator. THP induced damage to ³²P-labeled DNA fragments in the presence of metals. In the presence of Fe(III)EDTA, THP caused DNA damage at every nucleotide. The DNA damage was inhibited by free hydroxy radical (^·OH) scavengers and catalase, suggesting that the Fe(III)EDTA-mediated DNA damage is mainly due to ^·OH generation. In the presence of Cu(II), THP caused DNA damage mainly at T and G of 5′-TG-3′ sequence. The inhibitive effect of catalase and bathocuproine on Cu(II)-mediated DNA damage suggested that H₂O₂ and Cu(I) participate in the DNA damage. This study demonstrated that THP-induced apoptosis via reactive oxygen species generated from reaction of H₂O₂ and metals plays an important role in cytotoxicity of L-DOPA.     

Trimethylaurintricarboxylic acid inhibits human DNA methyltransferase 1: insights from enzymatic and molecular modeling studies

DNA methyltransferase 1 (DNMT1) is an emerging target for the treatment of cancer, brain disorders, and other diseases. Currently, there are only a few DNMT1 inhibitors with potential application as therapeutic agents or research tools. 5,5-Methylenedisalicylic acid is a novel scaffold previously identified by virtual screening with detectable although weak inhibitory activity of DNMT1 in biochemical assays. Herein, we report enzyme inhibition of a structurally related compound, trimethylaurintricarboxylic acid (NSC97317) that showed a low micromolar inhibition of DNMT1 (IC₅₀ = 4.79 μM). Docking studies of the new inhibitor with the catalytic domain of DNMT1 suggest that NSC97317 can bind into the catalytic site. Interactions with amino acid residues that participate in the mechanism of DNA methylation contribute to the binding recognition. In addition, NSC97317 had a good match with a structure-based pharmacophore model recently developed for inhibitors of DNMT1. Trimethylaurintricarboxylic acid can be a valuable biochemical tool to study DNMT1 inhibition in cancer and other diseases related to DNA methylation.     

Transactive response DNA binding protein of 43/histone deacetylase 6 axis alleviates H 2O 2-induced retinal ganglion cells injury through inhibiting apoptosis and autophagy

Oxidative damage is believed to contribute to the pathogenesis of diabetic retinopathy (DR). The current study aimed to detect the effects of transactive response DNA binding protein of 43 (TDP-43) on cell damage induced by hydrogen peroxide (H₂O₂) in retinal ganglion cells (RGCs) and to investigate the molecular mechanisms involved in this process. We observed that TDP-43 was highly expressed in RGC-5 cells induced by H₂O₂, and that repression of TDP-43 obviously ameliorated H₂O₂-induced RGC-5 cell injury. In addition, loss of TDP-43 profoundly mitigated H₂O₂-triggered oxidative stress by decreasing the production of intracellular reactive oxygen species and the activity of oxidative stress indicator malondialdehyde, as well as enhancing the content of antioxidant enzymes superoxide dismutase, glutathione peroxidase and catalase to restore the antioxidant defense system. Moreover, suppression of TDP-43 obviously obstructed H₂O₂-induced apoptosis. Meanwhile, knockdown of TDP-43 attenuated the expression of the proapoptotic proteins Bax and Cytochrome c, elevated the anti-apoptotic protein Bcl-2, and suppressed the activation of caspase 3 in H₂O₂-induced RGC-5 cells. Moreover, elimination of TDP-43 inhibited H₂O₂-triggered autophagy, which appeared as decreased expression of LC3II/I and Beclin-1, along with p62 degradation. Importantly, silencing of TDP-43 diminished the expression of histone deacetylase 6 (HDAC6), and HDAC6 also abolished the inhibitory effect of TDP-43 inhibition on H₂O₂-induced apoptosis and autophagy. Collectively, our findings demonstrated that depletion of TDP-43 may protect RGC-5 cells against oxidative stress-mediated apoptosis and autophagy by suppressing its target HDAC6. Thus, the TDP-43/HDAC6 axis might be a promising strategy for the treatment of DR.     

Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H₂O₂. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H₂O₂ levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H₂O₂ production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H₂O₂ must be taken into account in interpreting effects of phenolic compounds on cells in culture.