Saurabh Singh

Putative Mechanism for Anticancer and Apoptosis-Inducing Properties of Plant-Derived Polyphenolic Compounds

Several plant‐derived polyphenolic compounds are considered to possess anticancer and apoptosis‐inducing properties in cancer cells. Such compounds are recognized as naturally occurring antioxidants but also exhibit prooxidant properties under appropriate conditions. Evidence in the literature suggests that the antioxidant properties of polyphenolics such as gallotannins, curcumin, and resveratrol may not fully account for their chemopreventive effects. We propose a mechanism for the cytotoxic action of these compounds against cancer cells that involves mobilization of endogenous copper and the consequent prooxidant action.

Prooxidant and antioxidant activities of bilirubin and its metabolic precursor biliverdin: a structure–activity study

Bilirubin, which is derived from its metabolic precursor biliverdin, is the end product of heme catabolism. It has been proposed as a physiological antioxidant present in human extracellular fluids. We have earlier shown that bilirubin in the presence of the transition metal ion Cu(II) causes strand cleavage in DNA through generation of reactive oxygen species, particularly the hydroxyl radical. Thus bilirubin possesses both antioxidant and prooxidant properties. In order to understand the chemical basis of various biological properties of bilirubin, we have studied the structure-activity relationship between bilirubin and its precursor biliverdin. The latter has also been reported to possess both antioxidant and toxic properties. In the present studies bilirubin was found to be more effective in the DNA cleavage reaction and a more efficient reducer of Cu(II). The rate of formation of hydrogen peroxide and hydroxyl radicals by the compounds also showed a similar pattern. The relative antioxidant activity was also examined by studying the effect of these compounds on DNA cleavage by a hydroxyl radical generating system and their quenching effect on hydroxyl radicals. The results indicate that bilirubin is more active both as an antioxidant as well as an oxidative DNA cleaving agent. A model for binding of copper to bilirubin has been proposed where two copper ions are bound to two molecules of bilirubin through their terminal pyrrole nitrogens. In order to account for the enhanced copper reducing capacity of bilirubin we have further proposed that an additional copper binding site is provided for in the case of bilirubin due to the absence of a double bond between pyrrole rings II and III. Further it would appear that the structural features of the bilirubin molecule which are important for its prooxidant action are also the ones that render it a more effective antioxidant.

Bilirubin-Cu(II) complex degrades DNA

It has recently been reported that bilirubin forms a complex with Cu(II). In this paper we show that the formation of the complex results in the reduction of Cu(II) to Cu(I) and the redox cycling of the metal gives rise to the formation of reactive oxygen species, particularly hydroxyl radical. The bilirubin-Cu(II) complex causes strand breakage in calf thymus DNA and supercoiled plasmid DNA. Cu(I) was shown to be an essential intermediate in the DNA cleavage reaction by using the Cu(I) specific sequestering reagent neocuproine. Bilirubin-Cu(II) produced hydroxyl radical and the involvement of active oxygen species was established by the inhibition of DNA breakage by various oxygen radical quenchers.

Novel regulatory function for NHERF-1 in Npt2a transcription

Several lines of evidence show that sodium/hydrogen exchanger regulatory factor 1 (NHERF-1) regulates the expression and activity of the type IIa sodium-dependent phosphate transporter (Npt2a) in renal proximal tubules. We have previously demonstrated that expression of a COOH-terminal ezrin binding domain-deficient NHERF-1 in opossum kidney (OK) cells decreased expression of Npt2a in apical membranes but did not affect responses to parathyroid hormone. We hypothesized that NHERF-1 regulates apical membrane expression of Npt2a in renal proximal tubule cells. To address this hypothesis, we compared regulation of Npt2a expression and function in NHERF-deficient OK cells (OK-H) and wild-type cells (OK-WT). In OK-H cells, phosphate uptake and expression of Npt2a protein in apical membranes were significantly lower than in OK-WT cells. Transient transfection of green fluorescent protein-tagged Npt2a cDNA into OK-H cells resulted in aberrant localization of an Npt2a fragment to the cytosol but not to the apical membrane. OK-H cells also exhibited a marked decrease in Npt2a mRNA expression. As demonstrated by luciferase assay, Npt2a promoter activity was significantly decreased in OK-H cells compared with that shown in OK-WT cells. Transfection of OK-H cells with human NHERF-1 restored Npt2a expression at both the protein and mRNA levels and regulation by parathyroid hormone. Expression of NHERF-1 constructs with mutations in the PDZ domains or the ezrin binding domain in OK-H cells suggested that the PDZ2 domain is critical for apical translocation of Npt2a and for expression at the mRNA level. Our data demonstrate for the first time that NHERF-1 regulates Npt2a transcription and membrane insertion.

Bilirubin/biliverdin-Cu(II) induced DNA breakage; reaction mechanism and biological significance

Bilirubin and its metabolic precursor biliverdin are heme degradation products but have been proposed as physiological antioxidants. Reports from another laboratory as well as from ours have shown bilirubin to form a complex with the transition metal ion-Cu(II). Such a complex was shown by us to cause oxidative DNA damage. Further, biliverdin was also shown to be capable of causing similar DNA damage. In the present studies we have aimed to elucidate the mechanism of DNA breakage reaction by these bile pigments. Absorption and fluorescence studies indicate binding of bile pigments to DNA and copper ions. Cu(II) is reduced by these compounds to Cu(I) which is an essential intermediate in the DNA breakage reaction. Redox recycling of Cu(II) leads to generation of reactive oxygen species. Strand scission by the bile pigments-Cu(II) system is found to be biologically significant as assayed by bacteriophage inactivation. Our results, therefore are suggestive of one of the mechanisms through which endogenous DNA damage may occur.

Uric acid inhibits L-DOPA-CU(II) mediated DNA cleavage.

It has been proposed that considerable DNA damage may be caused by endogenous metabolites produced during the bodys normal metabolic processes. We have previously shown that L-DOPA, in the presence of Cu(II) leads to oxidative DNA breakage in vitro. Uric acid is considered to be a naturally occuring antioxidant and is present in plasma at a relatively high concentration. In this paper we report that uric acid inhibits L-DOPA-Cu(II) mediated DNA cleavage at concentrations similar to or lower than those found in plasma. Xanthine, which is the structural analogue of uric acid is a more potent inhibitor of the reaction. Uric acid was also shown to directly quench the generation of hydroxyl radicals by L-DOPA-Cu(II). The results have been discussed in relation to the putative protective role of uric acid against endogenous DNA damage by oxygen radicals.

Strand scission in DNA induced by 5-hydroxytryptamine (Serotonin) in the presence of copper ions

It has been proposed that considerable DNA damage may be caused by endogenous metabolites produced during the bodys normal metabolic processes. Several metabolites including L-3,4-dihydroxyphenylalanine and dopamine have been previously shown to lead to oxidative DNA breakage in the presence of copper ions. 5-hydroxytryptamine or serotonin is an important neurotransmitter in brain and spinal cord and is involved in the control of sleep and consciousness, aggression and mood. In this paper we show that serotonin is also capable of causing strand cleavage in DNA in the presence of Cu(II) through an oxidative mechanism. Serotonin reduces Cu(II) to Cu(I) which is accompanied by the generation of hydroxyl radical. Spectroscopic data indicate that serotonin is able to bind to DNA and copper ions. Relative DNA cleavage efficiency and copper binding ability of serotonin and structurally related molecules tryptophan and melatonin indicates that the phenolic group in serotonin is required for strand cleavage activity.

Inhibition of l-DOPA–Cu(II)-mediated DNA cleavage by bilirubin

It has been proposed that considerable DNA damage may be caused by endogenous metabolites produced during the bodys normal metabolic processes. We have previously shown that L-DOPA, in the presence of Cu(II), leads to oxidative DNA breakage in vitro. Bilirubin is a degradation product of heme and is considered to possess antioxidant properties. In this paper we report that bilirubin inhibits L-DOPA-Cu(II)-mediated DNA cleavage to an appreciable extent of 50% at a concentration of 50 microM. Bilirubin was also shown to directly quench the generation of hydroxyl radicals by L-DOPA-Cu(II) to an extent of 30% at a concentration of 20 microM. The results support the putative antioxidant role of bilirubin in higher primates.