Molecular beacon probes for the detection of cisplatin-induced DNA damage

Cisplatin (cis-diamminedichloroplatinum(II)) causes crosslinking of DNA at AG and GG sites in cellular DNA, inhibiting replication, and making it a useful anti-cancer drug. Several techniques have been used previously to detect nucleic acid damage but most of these tools are labour-intensive, time-consuming, and/or expensive. Here, we describe a sensitive, robust, and quantitative tool for detecting cisplatin-induced DNA damage by using fluorescent molecular beacon probes (MB). Our results show a decrease of fluorescence in the presence of cisplatin-induced DNA damage, confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The decrease in fluorescence upon damage scales with the number of AG and GG sites, indicating the ability of MB to quantitatively detect DNA damage by cisplatin.     

alpha-Mangostin enhances betulinic acid cytotoxicity and inhibits cisplatin cytotoxicity on HCT 116 colorectal carcinoma cells

Despite the progress in colon cancer treatment, relapse is still a major obstacle. Hence, new drugs or drug combinations are required in the battle against colon cancer. α-Mangostin and betulinic acid (BA) are cytotoxic compounds that work by inducing the mitochondrial apoptosis pathway, and cisplatin is one of the most potent broad spectrum anti-tumor agents. This study aims to investigate the enhancement of BA cytotoxicity by α-mangostin, and the cytoprotection effect of α-mangostin and BA on cisplatin-induced cytotoxicity on HCT 116 human colorectal carcinoma cells. Cytotoxicity was investigated by the XTT cell proliferation test, and the apoptotic effects were investigated on early and late markers including caspases-3/7, mitochondrial membrane potential, cytoplasmic shrinkage, and chromatin condensation. The effect of α-mangostin on four signalling pathways was also investigated by the luciferase assay. α-Mangostin and BA were more cytotoxic to the colon cancer cells than to the normal colonic cells, and both compounds showed a cytoprotective effect against cisplatin-induced cytotoxicity. On the other hand, α-mangostin enhanced the cytotoxic and apoptotic effects of BA. Combination therapy hits multiple targets, which may improve the overall response to the treatment, and may reduce the likelihood of developing drug resistance by the tumor cells. Therefore, α-mangostin and BA may provide a novel combination for the treatment of colorectal carcinoma. The cytoprotective effect of the compounds against cisplatin-induced cytotoxicity may find applications as chemopreventive agents against carcinogens, irradiation and oxidative stress, or to neutralize cisplatin side effects.     

The Proteins from Sika deer antler as potential modulators on cisplatin-induced cytotoxicity in human embryonic kidney 293 cells

Our study aimed to investigate the protective role of SDAPR on cisplatin-induced cytotoxicity and its’ possible mechanism in HEK293 cells. Cell viability was measured by MTT assay. Oxidative stress (SOD, GSH, LDH and MDA), inflammatory factors (TNF-α and IL-6) and apoptosis-related proteins (caspase-3, Bax, Bcl-2) expression were measured. The apoptotic cells were observed by TUNEL staining. Our study results indicated that non-cytotoxic levels of SDAPR significantly increased viability rate (LD₅₀ value of cisplatin is 20 μM), which improved antioxidant defence, attenuated apoptosis by decreasing expression levels of cleaved-caspase-3 and Bax, increasing Bcl-2 expression and inhibiting apoptotic positive cells in HEK 293 cells. In addition, SDAPR treatment markedly inhibited the levels of TNF-α and IL-6. In conclusion, Sika deer antler protein, a potential modulator, could alleviate cisplatin-induced cytotoxicity in HEK 293 cells.     

DNA damage by fasicularin

Fasicularin is a structurally novel thiocyanate-containing alkaloid isolated from the ascidian Nephteis fasicularis. Early biological experiments suggested that this compound's cytotoxic properties may stem from its ability to damage cellular DNA. Sequence gel analysis reveals that treatment of a 5'-32P-labeled DNA duplex with fasicularin in pH 7.0 buffer causes strand cleavage selectively at guanine residues. Further experiments indicate that production of these base-labile lesions in DNA involves alkylation of guanine residues by a fasicularin-derived aziridinium ion. This work reveals fasicularin as the first natural product found to generate a DNA-alkylating aziridinium ion via a mechanism analogous to the clinically used anticancer drugs mechlorethamine, melphalan, and chlorambucil.     

Millettia ferruginea extract attenuates cisplatin-induced alterations in kidney functioning,DNA damage,oxidative stress,and renal tissue morphology

This study aimed to investigate the beneficial role of Millettia ferruginea extract (MF) in preventing cisplatin (Cisp) induced nephrotoxicity in rats. A total of 55 metabolites were identified using LC-MS analysis. The in vivo results indicated that MF pretreatment for 4 weeks (20 mg/kg b.w.) remarkably attenuated the altered renal biomarkers by decreasing the levels of plasma creatinine, urea, and uric acid when compared to the Cisp-group. The nephroprotective capacity of MF was further strengthened by histopathological observations, where Cisp + MF treated rats showed lower number of inflammatory cells and tubular degenerative changes than the Cisp-group. The harmful effects of cisplatin on renal oxidative stress indicators (MDA, SOD, CAT, and GPx), were restored by the treatment of MF. In addition, the reduction of inflammatory markers (IL-6 and TNF-α), associated with alleviating DNA fragmentation, highlighted the preventive effect of MF in kidney tissue. Additionally, MF components presented lower binding energies when docked into the active site of TNF-α and IL-6. The present findings concluded that M. ferruginea extract exhibited nephroprotective potential, which may be attributed to its antioxidant and anti-inflammatory properties. Further work is recommended to confirm the current results, explore the involved mechanism of action, and determine the therapeutic doses and time.     

Temperature-dependent formation and photorepair of DNA damage induced by UV-B radiation in suspension-cultured tobacco cells

Two photoproducts of DNA damage, i.e. cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs), induced by UV-B radiation in suspension-cultured tobacco cells were quantified by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. CPDs and 6-4PPs were induced in tobacco cells by UV-B radiation. Photorepair of CPDs was faster than that of 6-4PPs. UV-B radiation induces formation of CPDs and 6-4PPs even at 0 degrees C, but low temperature significantly decreases the UV-B-induced (in contrast to UV-C-induced) formation of CPDs and 6-4PPs. Low temperature also retarded the removal of CPDs and 6-4PPs under white light, and almost no photorepair of CPDs and 6-4PPs was detected at 0 degrees C. When purified DNA from tobacco cells grown in darkness was irradiated with UV-B, formation of CPDs and 6-4PPs took place at the same speed at different temperatures. It indicated that formation of CPDs and 6-4PPs induced by UV-B was temperature-independent in a non-cellular system. Based on our results for suspension-cultured tobacco cells, not only the photorepair but also UV-B-induced formation of CPDs and 6-4PPs are temperature-dependent.     

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.     

Analysis of metal-induced oxidative DNA damage in cultured mammalian cells

Reactive oxygen species are continuously generated during oxygen metabolism, and a measurable amount of oxidative DNA damage exists in aerobic organisms. By the determination of Fpg-sensitive sites in mammalian cells in culture, we assessed the background level of oxidative DNA damage and its potential increase by extracellularly applied complexes of iron(III). In V79 Chinese hamster cells the endogenous level of Fpg-sensitive modifications is detectable, but the extent is much lower as compared with results derived from other analytical methods. In V79 cells, the frequency of Fpg-sensitive modifications is considerably enhanced by Fe-NTA in a time- and dose-dependent manner, while no increase is observed after treatment with Fe-citrate. These results indicate that the ability of transition metals to generate oxidative DNA damage in intact cells strongly depends on factors like uptake and intracellular distribution, which will affect the intracellular availability of redox-active metal ions close to critical targets.     

Efficient photoinduced DNA damage by coralyne

The photoinduced DNA damage by the berberine derivative coralyne is presented. The irradiation of coralyne in the presence of plasmid DNA namely, pBR322, leads to remarkably fast DNA damage by single-strand cleavage, as determined by agarose-gel electrophoresis. Even upon exposure to sunlight, almost all of the supercoiled plasmid is converted to the open circular form in less than a minute [c(pBR322) = 3.5 x 10(-9) M; c(coralyne) = 4.3 x 10(-5) M]. The efficiency of the DNA strand cleavage is not decreased in the presence of radical-trapping reagents such as tert-butanol or DMSO. Moreover, the extent of the DNA damage is the same under aerobic conditions and at reduced oxygen concentration. Thus, the formation of reactive intermediates such as hydroxyl radicals or singlet oxygen is excluded. These results show that the exposure of coralyne and derivatives thereof to light, even with moderate light intensity, needs to be avoided during experiments in which their biological activity is assessed by plasmid unwinding assays.     

Analysis of UV-B-induced DNA damage and its repair in heat-shocked skin cells

The heat-shock response is a cellular defence mechanism against environmental stresses that is evolutionarily conserved from bacteria to man. Numerous reports demonstrate the beneficial effects of heat-shock protein induction on cell survival under toxic or oxidative stress, e.g., in cardiac and cerebral ischemia or prior to organ transplantation. However, there is little data on the effects of heat treatment on damage caused by UV irradiation. Applying three independent techniques, we have tested the influence of thermal pretreatment of skin cells (1 h, 43 degrees C) on the initial extent of UV-B-induced DNA damage and its subsequent repair. For cultured human epidermal keratinocytes and dermal fibroblasts we can show reduced levels of nucleotide-excision-repair-associated DNA strand incision in the comet assay. Moreover, immunostaining and flow cytometric quantitation of thymidine dimers immediately and one day after irradiation, respectively, reveal that the initial DNA damage is not (keratinocytes) or only moderately (fibroblasts) lower in heat-shocked cells as compared to untreated controls. However, excision repair of dimers is significantly attenuated during the first 24 h in both cell types. Furthermore, using a modified host-cell reactivation assay, we are able to demonstrate that repair of UV-B-damaged plasmid DNA is lower if the transfected cells are previously heat shocked. In summary, heat treatment (1 h, 43 degrees C) inducing heat-shock proteins reduces nucleotide excision repair of UV-B-mediated DNA lesions in fibroblasts and keratinocytes during the following 24 h. This is not necessarily caused by elevated heat-shock protein levels themselves. Possibly the direct thermal damage of repair enzymes is more severe than the potential protective effects of heat-shock proteins.     

Parthenolide induces apoptosis and cell cycle arrest of human 5637 bladder cancer cells in vitro

Parthenolide, the principal component of sesquiterpene lactones present in medical plants such as feverfew (Tanacetum parthenium), has been reported to have anti-tumor activity. In this study, we evaluated the therapeutic potential of parthenolide against bladder cancer and its mechanism of action. Treatment of bladder cancer cells with parthenolide resulted in a significant decrease in cell viability. Parthenolide induced apoptosis through the modulation of Bcl-2 family proteins and poly (ADP-ribose) polymerase degradation. Treatment with parthenolide led to G1 phase cell cycle arrest in 5637 cells by modulation of cyclin D1 and phosphorylated cyclin-dependent kinase 2. Parthenolide also inhibited the invasive ability of bladder cancer cells. These findings suggest that parthenolide could be a novel therapeutic agent for treatment of bladder cancer.     

DNA damage in nucleosomes

Eukaryotic genomic DNA is packed into chromatin, whose fundamental structural unit is the nucleosome. As DNA-histone protein complexes, nucleosomes show different properties toward exogenous and endogenous DNA-damaging agents. This review summarizes nucleosome DNA damage due to different sources, including alkylating agents, radicals, UV radiation and reactive DNA damage intermediates. In most cases, the histone core protects the associated DNA against damage via its structure and/or scavenging of damaging agents. In contrast, histones react with damaged DNA and, in some instances, catalyze DNA damage in the nucleosome. The biological consequence of nucleosome DNA damage and future prospects in this field are briefly discussed.     

UVA-induced oxidative damage and cytotoxicity depend on the mode of exposure

The reciprocity rule (Bunsen-Roscoe law) states that a photochemical reaction is directly proportional to the total energy dose, irrespective of the dose distribution. In photomedicine the validity of this law is usually taken for granted, although the influence of radiation intensity and dose distribution are largely unknown. We have examined in a tissue culture model the effects of fractionated versus single dose exposure to UV from a metal halide source on survival, DNA synthesis, glutathione, and oxidative membrane damage. Exposure to fractionated UVA was followed by an increased rate of cell death compared to single dose exposure, when intervals between fractions where short (10-120 min). Longer intervals had the opposite effect. Corresponding results were obtained for DNA synthesis (BrdU incorporation). The increased cytotoxicity of dose fractionation with short intervals could not be abrogated by non-enzymatic antioxidants (astaxanthin, ascorbic acid, alpha-tocopherol). Fractionated irradiation with short intervals led to higher degree of depletion of glutathione (GSH) and to enhanced formation of thiobarbituric acid reactive substances (TBARS) in comparison to an identical single dose. Long intervals between fractions induced opposite effects. Taken together, these data indicate that immediately after UVA exposure cells are more sensitive to a further oxidative attack making repeated exposure with short intervals more cytotoxic than continuous single dose UVA. This might have implications also for responses to UVA in vivo and further studies will have to extend these findings to the situation in healthy and diseased human skin.     

Increasing the speed limit for hole transport in DNA

Transport of positive charge or holes in DNA occurs via a thermally activated multi-step hopping mechanism. The fastest hopping rates reported to date are those for repeating poly(purine) sequences in which hopping occurs via a random walk mechanism with rate constants of k(hop) = 4.3 × 10(9) s(-1) for poly(dG) and 1.2 × 10(9) s(-1) for poly(dA). We report here the dynamics of charge separation in DNA conjugates possessing repeating 7-deazaadenine (dzA) sequences. These data provide an estimated value of k(hop) = 4.2 × 10(10) s(-1) for poly(dzA), an order of magnitude faster than for poly(dG).     

DNA damage by sulfite autoxidation catalyzed by cobalt complexes

DNA damage was investigated in the presence of sulfite, dissolved oxygen and cobalt(II) complexes with glycylglycylhistidine, glycylhistidyllysine, glycylglycyltyrosylarginine and tetraglycine. These studies indicated that only Co(II) complexed with glycylglycylhistidine (GGH) induced DNA strand breaks at low sulfite concentrations (1-80 microM) via strong oxidants formed in the reaction. In the presence of the other complexes, some damage occurred only in the presence of high sulfite concentrations (0.1-2.0 mM) after incubation for 4 h. In the presence of GGH, Co(II) and dissolved O2, DNA damage must involve a reactive high-valent cobalt complex. The damaging effect was increased by adding S(IV), due to the oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by the complex. SO3 -, HO and H radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline N-oxide). The results indicate that Co(II) binds O2 in the presence of GGH, and leads to the formation of a DMPO-HO adduct without first forming free superoxide or hydroxyl radical, supporting the participation of a reactive high-valent cobalt complex.     

Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to function optimally under one condition only. Microarrays are often burdened with a significant degree of cross-hybridization, because of a poor combination of assay conditions and probe choice. As reviewed here, a number of promising microfluidics-based technologies can provide automatic processing of arrays under different assay conditions. These new array processors provide researchers and assay developers with novel possibilities to construct highly specific DNA arrays even towards regions of DNA greatly varying in G?+?C content. These array processors are also a powerful development tool for building arrays, because they combine high sample throughput with investigation of optimal assay conditions. The array processors can increase specificity in all DNA microarray assays, e.g. for gene expression, and microRNA and mutation analysis. Increased specificity of the array will also benefit microarray-based loci selection prior to high-throughput sequencing.     

Targets of red grapes: oxidative damage of DNA and leukaemia cells

Vitis vinifera is a widespread crop all over the world. The biophenols present in grapes have a remarkable influence on wine quality and also confer potential health-protecting properties to this fruit. The aim of the present work was to assess the beneficial properties of skin, seeds and pulp of red table grapes (RTG) (Vitis vinifera, Palieri Cadiz variety). Two potential beneficial activities of red table grapes (RTG) were analyzed: (i) The maintenance of genomic stability studying their genotoxic/antigenotoxic effects, and (ii) the in vitro cytotoxic effect against tumor cells of RTG components. The genotoxic and/or antigenotoxic effect was measured applying the somatic mutation and recombination test on wing imaginal discs of Drosophila melanogaster. The cytotoxic effect was monitored using the HL60 human leukemia model to evaluate the antiproliferative potential of the different parts of RTG. The three major parts (skin, seeds and pulp) are not genotoxic. When antigenotoxicity assays were performed using hydrogen peroxide as the oxidative genotoxin, skin, seed and pulp exerted a desmutagenic effect, with seeds and skin showing the most potent effect. The cytotoxicity tests using HL60 cells indicated that only skin and pulp fractions are able to inhibit the tumor growth, with skin having the lowest IC50 (1.8 mg/mL versus 8 mg/mL of pulp). These results suggest that RTG are potent anti-mutagens that protect DNA from oxidative damage as well as being cytotoxic toward the HL60 tumor cell line.     

Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species

The purpose of this study was to evaluate the ability of the predominant carotenoids (lutein and zeaxanthin) of the macular pigment of the human retina, to protect SK-N-SH human neuroblastoma cells against DNA damage induced by different RNOS donors. Although astaxanthin has never been isolated from the human eye, it was included in this study because its structure is very close to that of lutein and zeaxanthin and because it affords protection from UV-light. DNA damage was induced by GSNO-MEE, a nitric oxide donor, by Na(2)N(2)O(3), a nitroxyl anion donor and by SIN-1, a peroxynitrite-generating agent. DNA damage was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique able to detect primary DNA damage in individual cells. The tail moment parameter was used as an index of DNA damage. The values of tail moment increased in all the samples incubated with the RNOS donors, indicating DNA impairment. Data obtained show that the ability of zeaxanthin, lutein, and astaxanthin to reduce the DNA damage depends on the type of RNOS donor and the carotenoid concentration used. All the carotenoids studied were capable of protecting against DNA damage in neuroblastoma cells when the cells were exposed to GSNO-MEE. However, a different behaviour was present when the other two RNOS donors were used. The presence of a carotenoid alone (without an RNOS donor) did not cause DNA damage. Spectrophotometric studies showed that the order with which tested carotenoids reacted with RNOS was not always in agreement with the DNA protection results. The data from this study provides additional information on the activities of the macular pigment carotenoids of the human retina.     

Influence of astaxanthin, zeaxanthin and lutein on DNA damage and repair in UVA-irradiated cells

In order to gain more knowledge about the antioxidant role of the predominant carotenoids (lutein and zeaxanthin) of the human retina, this study investigated their antioxidant activity and capacity. Astaxanthin was also studied, because its structure is very close to that of lutein and zeaxanthin. The antioxidant activity of these molecules was evaluated using chemiluminescence techniques, with lucigenin and luminol as chemiluminogenic probes for the superoxide radical and hydrogen peroxide, respectively. It was found that all three carotenoids have similar superoxide-scavenging activity. The effect on the reduction of H(2)O(2)-luminol chemiluminescence was present in the following order, zeaxanthin>astaxanthinlutein. Possible antioxidant capacity of these three compounds was sought using a biological system consisting of SK.N.SH human neuroblastoma and rat trachea epithelial cells subjected to oxidative stress from exposure to UVA radiation. In particular, we determined whether these compounds were capable of minimizing DNA damage and influencing the kinetics of DNA repair. DNA damage was assessed using the Comet assay, a rapid and sensitive single-cell gel electrophoresis technique used to detect primary DNA damage in individual cells. Neuroblastoma cells appeared more resistant to oxidative irradiation insult. The presence of carotenoids reduced DNA damage when rat epithelial cells were exposed to UVA radiation for 2min. A different result was obtained in experiments performed on neuroblastoma cells; in this case, the presence of carotenoid during UVA exposition increased the damage. The addition of carotenoids to epithelial cells after 2min of UVA exposition did not seem to improve the kinetics of DNA repair; on the contrary, zeaxanthin (after 60' incubation) and lutein (after 180' incubation) showed a genotoxic effect. The addition of carotenoids to neuroblastoma cells after 30' UVA exposition positively influences the kinetics of DNA repair in the first 15min of incubation. At longer exposition times, while the behaviour measured was not constant, a genotoxic effect was not observed. The data from this study provide additional information on the antioxidant and pro-oxidant activities of the predominant macular pigment carotenoids of the human retina.