Catechin Photolysis Suppression by Aluminum Chloride under Alkaline Conditions and Assessment with Liquid Chromatography-Mass Spectrometry.

Tea is rich in catechins and aluminum. In this study, the process of catechin photolysis was applied as a model for examining the effects of aluminum chloride (AlCl3) on the structural changes of catechin and the alteration of aluminum complexes under blue light irradiation (BLI) at pH 8 using liquid chromatography and mass spectrometry techniques. Additionally, the effects of anions on catechin upon the addition of AlCl3 and treatment with BLI were also studied. In this study, when 1 mM catechin was treated with BLI, a superoxide anion radical (O2•-) was generated in an air-saturated aqueous solution, in addition to forming a dimeric catechin (proanthocyanidin) via a photon-induced redox reaction. The relative percentage of catechin was found to be 59.0 and 95.7 for catechin treated with BLI and catechin upon the addition of 1 mM AlCl3 treated with BLI, respectively. It suggested that catechin treated with BLI could be suppressed by AlCl3, while AlCl3 did not form a complex with catechin in the photolytic system. However, under the same conditions, it was also found that the addition of AlCl3 inhibited the photolytic formation of O2•-, and reduced the generation of proanthocyanidin, suggesting that the disconnection of proanthocyanidin was achieved by AlCl3 acting as a catalyst under treatment with BLI. The influence of 1 mM fluoride (F-) and 1 mM oxalate (C2O42-) ions on the photolysis of 1 mM catechin upon the addition of 1 mM AlCl3 and treatment with BLI was found to be insignificant, implying that, during the photolysis of catechin, the Al species were either neutral or negatively charged and the aluminum species did not form a complex with anions in the photolytic system. Therefore, aluminum, which is an amphoteric species, has an inherent potential to stabilize the photolysis of catechin in an alkaline conditions, while suppressing the O2•- and proanthocyanidin generation via aluminum ion catalysis in the catechin/Al system under treatment with BLI.

Effects of Epigallocatechin Gallate on the Stability of Epicatechin in a Photolytic Process.

Catechins belonging to polyhydroxylated polyphenols are the primary compounds found in green tea. They are associated with many physiological properties. Epicatechin (EC) is a non-gallate-type catechin with four phenolic hydroxyl groups attached. The changes in EC treated with color light illumination in an alkaline condition were investigated by chromatographic and mass analyses in this study. In particular, the superoxide anion radical (O2•-) was investigated during the EC photolytic process. EC is unstable under blue light illumination in an alkaline solution. When EC was treated with blue light illumination in an alkaline solution, O2•- was found to occur via a photosensitive redox reaction. In addition, the generation of monomeric, dimeric, and trimeric compounds is investigated. On the other hand, epigallocatechin gallate (EGCG), which is a gallate-type catechin, is stable under blue light illumination in an alkaline solution. Adding EGCG, during the blue light illumination treatment of EC decreased photolytic formation, suggesting that gallate-type catechins can suppress the photosensitive oxidation of EC. Gallate-type catechins are formed via the esterification of non-gallate-type catechins and gallic acid (GA). The carbonyl group on the gallate moiety of gallate-type catechins appears to exhibit its effect on the stability against the photosensitive oxidation caused by blue light illumination.

Effects of Blue-Light-Induced Free Radical Formation from Catechin Hydrate on the Inactivation of Acinetobacter baumannii, Including a Carbapenem-Resistant Strain.

Catechin is a flavan-3-ol, a derivative of flavans, with four phenolic hydroxyl groups, which exhibits a wide range of physiological properties. Chromatographic analyses were employed to examine the effects of blue light irradiation on the changes of catechin hydrate in an alkaline condition. In particular, the detection of a superoxide anion radical (O2•−), a reactive oxygen species (ROS), and the inactivation of Acinetobacter baumannii (A. baumannii)­including a carbapenem-resistant A. baumannii (CRAB)­was investigated during the photoreaction of catechin hydrate. Following basification with blue light irradiation, the transparent solution of catechin hydrate turned yellowish, and a chromogenic catechin dimer was separated and identified as a proanthocyanidin. Adding ascorbic acid during the photolytic treatment of catechin hydrate decreased the dimer formation, suggesting that ascorbic acid can suppress the photosensitive oxidation of catechin. When catechin hydrate was irradiated by blue light in an alkaline solution, O2•− was produced via photosensitized oxidation, enhancing the inactivation of A. baumannii and CRAB. The present findings on the photon-induced oxidation of catechin hydrate provides a safe practice for the inactivation of environmental microorganisms.

Crystal structure-based exploration of the important role of Arg106 in the RNA-binding domain of human coronavirus OC43 nucleocapsid protein.

Human coronavirus OC43 (HCoV-OC43) is a causative agent of the common cold. The nucleocapsid (N) protein, which is a major structural protein of CoVs, binds to the viral RNA genome to form the virion core and results in the formation of the ribonucleoprotein (RNP) complex. We have solved the crystal structure of the N-terminal domain of HCoV-OC43 N protein (N-NTD) (residues 58 to 195) to a resolution of 2.0Å. The HCoV-OC43 N-NTD is a single domain protein composed of a five-stranded ß-sheet core and a long extended loop, similar to that observed in the structures of N-NTDs from other coronaviruses. The positively charged loop of the HCoV-OC43 N-NTD contains a structurally well-conserved positively charged residue, R106. To assess the role of R106 in RNA binding, we undertook a series of site-directed mutagenesis experiments and docking simulations to characterize the interaction between R106 and RNA. The results show that R106 plays an important role in the interaction between the N protein and RNA. In addition, we showed that, in cells transfected with plasmids that encoded the mutant (R106A) N protein and infected with virus, the level of the matrix protein gene was decreased by 7-fold compared to cells that were transfected with the wild-type N protein. This finding suggests that R106, by enhancing binding of the N protein to viral RNA plays a critical role in the viral replication. The results also indicate that the strength of N protein/RNA interactions is critical for HCoV-OC43 replication.

Photodynamic and Antibacterial Assessment of Gold Nanoparticles Mediated by Gold (III) Chloride Trihydrate and Sodium Citrate under Alkaline Conditions.

Sodium citrate (SC) is sensitive to violet light illumination (VLI) and acts as a weak reductant. Conversely, gold (III) chloride trihydrate (GC) often acts as an oxidant in a redox reaction. In this study, the influences of colored light on the production of gold nanoparticles (AuNPs) in a mixture of gold (III) ions and citrate via VLI and the antibacterial photodynamic inactivation (aPDI) of Escherichia coli (E. coli) are determined under alkaline conditions. The diameter of AuNPs is within the range of 3-15 nm, i.e., their mean diameter is 9 nm; when citrate is mixed with gold (III) ions under VLI, AuNPs are formed via an electron transfer process. Additionally, GC mixed with SC (GCSC) inhibits E. coli more effectively under VLI than it does under blue, green, or red light. GCSC and SC are shown to inhibit E. coli populations by 4.67 and 1.12 logs, respectively, via VLI at 10 W/m2 for 60 min under alkaline conditions. GCSC-treated E. coli has a more significant photolytic effect on anionic superoxide radical (O2•-) formation under VLI, as more O2•- is formed within E. coli if the GCSC-treated samples are subjected to VLI. The O2•- exhibits a greater effect in a solution of GCSC than that shown by SC alone under VLI treatment. Gold (III) ions in a GCSC system appear to act as an oxidant by facilitating the electron transfer from citrate under VLI and the formation of AuNPs and O2•- via GCSC photolysis under alkaline conditions. As such, the photolysis of GCSC under VLI is a useful process that can be applied to aPDI.

The effect of photolysis of sodium citrate treated with gold chloride using coloured light on the generation of gold nanoparticles and the repression of WiDr colon cancer cells.

Gold nanoparticles (GNPs) are usually formed via a wet chemical method using gold (III) chloride trihydrate (GC), which is treated with stable reducing agents such as sodium citrate (SC). This study determines the effect of coloured light on the formation of GNPs by irradiation of SC after the addition of GC (SCGC) and the effect of the SCGC photolytic procedure on the suppression of WiDr colon cancer cells by forming reactive oxygen species. The absorbance of surface plasmon resonance peaks at 523 nm are 0.069 and 0.219 for SCGC when treated with blue light illumination (BLI) and violet light irradiation (VLI), respectively, whereas green and red light treatments have little or no effect. Most GNPs have diameters ranging from 3 to 15 nm, with a mean of 6 nm, when SCGC is exposed to VLI for 1.5 h. Anionic superoxide radicals (O2•-) are formed in a charge-transfer process after SCGC under VLI treatment; however, BLI treatment produces no significant reaction. Moreover, SCGC under VLI treatment proves to be considerably more effective at inhibiting WiDr cells than BLI treatment, as firstly reported in this study. The reduction rates for WiDr cells treated with SCGC under BLI and VLI at an intensity of 2.0 mW/cm2 for 1.5 h (energy dose, 10.8 J/cm2) are 4.1% and 57.7%, respectively. The suppression rates for WiDr cells treated with SCGC are inhibited in an irradiance-dependent manner, the inhibition percentages being 57.7%, 63.3%, and 80.2% achieved at VLI intensities of 2.0, 4.0, and 6.0 mW/cm2 for 1.5 h, respectively. Propidium iodide is a fluorescent dye that detects DNA changes after cell death. The number of propidium iodide-positive nuclei significantly increases in WiDr cells treated with SCGC under VLI, suggesting that SCGC photolysis under VLI is a potential treatment option for the photodynamic therapy process.

The effects of loop size on Sac7d-hairpin DNA interactions.

Hairpin structure is a common feature of DNA molecules. They are located near functional loci, such as regulation and promotion sites, as well as in cruciform structures, and they provide potential binding sites for endogenous proteins. The effects of different hairpin loops that are composed of one to five thymidines, designated as L1-L5, and have a common self-complementary stem, CTATATAG, on the interactions with Sac7d were studied. In thermostability studies, Sac7d stabilized a tetra-loop hairpin DNA and hairpin DNA with GTTC tetra-loop regions better than it stabilized tri- and penta-loops. Circular dichroism (CD) spectra showed that hairpins retained primarily a B-type conformation upon Sac7d binding. Intermolecular interactions between hairpins were likely decreased, due to the Sac7d-induced kinks, as shown by an increase at 220nm in the CD spectra. Surface plasmon resonance (SPR) observations suggested that the rates of Sac7d binding to hairpin DNA depend on the loop size of the hairpin duplexes. At a fixed stem length, Sac7d binds to tetra-loop hairpin DNA duplexes with a higher association rate and lower dissociation rate, compared with their tri- and penta-loop counterparts. In addition, the tri-loop and GTC tri-loop hairpin DNA had lower affinity for Sac7d because of the smaller and tighter loop size. Our study indicates that Sac7d binding affinity to hairpin DNA is primarily determined by loop size and stem integrity, and the results presented here provide a model for studies concerning other minor groove DNA-binding proteins that kink hairpin DNA.

An investigation of the influence of reactive oxygen species produced from riboflavin-5'-phosphate by blue or violet light on the inhibition of WiDr colon cancer cells.

Riboflavin-5'-phosphate (FMN), an innocuous product of riboflavin (RF) phosphorylation, is vital for humans. FMN is sensitive to light illumination, as indicated by reactive oxygen species (ROS) formation. This investigation was undertaken to evaluate the influence of blue light illumination (BLI) and violet light illumination (VLI) upon FMN to develop a method to inhibit WiDr colon cancer cells by FMN photolysis. When FMN is subjected to BLI and VLI, it inhibits WiDr colon cancer cells by generating superoxide radical anions (O2•-). The respective reduction rates are 42.6 and 81.9 % in WiDr colon cancer cells for FMN treated with BLI and VLI at 20 W/m2 for 0.5 h. FMN treated with VLI inhibits WiDr colon cancer cells more effectively than BLI. Propidium iodide (PI) is a fluorescent dye that is used to detect abnormal DNA due to cell death by apoptosis or necrosis. The PI-positive count for nuclei increased significantly for the WiDr colon cancer cells that were treated with FMN under VLI at 20 W/m2 for 0.5 h. FMN photolysis achieved using VLI allows efficient photodynamic therapy (PDT) by triggering the cytotoxicity of FMN on WiDr colon cancer cells.

Inactivation of Pathogens via Visible-Light Photolysis of Riboflavin-5'-Phosphate.

Riboflavin-5'-phosphate (or flavin mononucleotide; FMN) is sensitive to visible light. Various compounds, including reactive oxygen species (ROS), can be generated from FMN photolysis upon irradiation with visible light. The ROS generated from FMN photolysis are harmful to microorganisms, including pathogenic bacteria such as Staphylococcus aureus (S. aureus). This article presents a protocol for deactivating S. aureus, as an example, via photochemical reactions involving FMN under visible light irradiation. The superoxide radical anion () generated during the FMN photolysis is evaluated via nitro blue tetrazolium (NBT) reduction. The microbial viability of S. aureus that is attributed to reactive species was used to determine the effectiveness of the process. The bacterial inactivation rate is proportional to FMN concentration. Violet light is more efficient in inactivating S. aureus than blue light irradiation, while the red or green light does not drive FMN photolysis. The present article demonstrates FMN photolysis as a simple and safe method for sanitary processes.

A study of the effect of reactive oxygen species induced by violet and blue light from oxytetracycline on the deactivation of Escherichia coli.

Oxytetracycline (OTC), a tetracycline antibiotic, is a broad-spectrum antibacterial agent. In this investigation, liquid chromatography-mass spectrometry (LC-MS) is utilized to determine the effects of blue light (λ = 448 nm) illumination (BLIA) and violet light (λ = 403 nm) illumination (VLIA) on conformational changes in OTC at pH 7.8. The photochemical effect of OTC that is exposed to BLIA and VLIA on the deactivation of Escherichia coli (E. coli) is studied. The deactivation of E. coli has an insignificant effect on treatment with OTC alone. OTC is relatively unstable under BLIA and VLIA illumination in an alkaline solution, and OTC has been shown to inactivate E. coli by generating reactive oxygen species (ROS). Less anionic superoxide radicals (O2•-) are generated from OTC that is treated with BLIA than that from VLIA treatment, so OTC is more efficient in inactivating E. coli under VLIA. Inactivation of reduction rates of 0.51 and 3.65 logs in E. coli are achieved using 0.1 mM OTC under BLIA for 120 min and VLIA for 30 min, respectively, under the same illumination intensity (20 W/m2). Two photolytic products of OTC (PPOs) are produced when OTC is exposed to BLIA and VLIA, with molecular ions at m/z 447 and 431, molecular formulae C21H22N2O9 and C21H22N2O8, and masses of 446.44 and 430.44 g/mol, respectively. The results show that when exposed to VLIA, OTC exhibits enhanced inactivation of E. coli, suggesting that the photochemical treatment of OTC is a potential supplement in a hygienic process.

Effects of free radicals from doxycycline hyclate and minocycline hydrochloride under blue light irradiation on the deactivation of Staphylococcus aureus, including a methicillin-resistant strain.

Doxycycline hyclate (DCH) and minocycline hydrochloride (MH) are tetracycline antibiotics and broad-spectrum antimicrobial agents. The changes in DCH and MH under blue light (λ = 462 nm) irradiation in alkaline conditions (BLIA) were investigated. Deactivation caused by superoxide anion radical (O2•-) and deactivation from DCH and MH during photolysis on Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), were studied. DCH is relatively unstable compared to MH under BLIA. The level of O2•- generated from the MH-treated photoreaction is lower than that from DCH photolysis, and the DCH-treated photoreaction is more efficient at inactivating S. aureus and MRSA at the same radiant intensity. DCH subjected to BLIA decreased the viability of S. aureus and MRSA by 3.84 and 5.15 log, respectively. Two photolytic products of DCH (PPDs) were generated under BLIA. The mass spectra of the PPDs featured molecular ions at m/z 460.8 and 458.8. The molecular formulas of the PPDs were C21H22N2O10 and C22H24N2O9, and their exact masses were 462.44 and 460.44 g/mol, respectively. These results bolster the photolytic oxidation that leads to DCH-enhanced deactivation of S. aureus and MRSA. Photochemical treatment of DCH could be applied as a supplement in hygienic processes.

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% ß-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB.

Effects of polyamines on the DNA-reactive properties of dimeric mithramycin complexed with cobalt(II): implications for anticancer therapy.

Few studies have examined the effects of polyamines on the action of DNA-binding anticancer drugs. Here, a Co(II)-mediated dimeric mithramycin (Mith) complex, (Mith)(2)-Co(II), was shown to be resistant to polyamine competition toward the divalent metal ion when compared to the Fe(II)-mediated drug complexes. Surface plasmon resonance experiments demonstrated that polyamines interfered with the binding capacity and association rates of (Mith)(2)-Co(II) binding to DNA duplexes, while the dissociation rates were not affected. Although (Mith)(2)-Co(II) exhibited the highest oxidative activity under physiological conditions (pH 7.3 and 37 degrees C), polyamines (spermine in particular) inhibited the DNA cleavage activity of the (Mith)(2)-Co(II) in a concentration-dependent manner. Depletion of intracellular polyamines by methylglyoxal bis(guanylhydrazone) (MGBG) enhanced the sensitivity of A549 lung cancer cells to (Mith)(2)-Co(II), most likely due to the decreased intracellular effect of polyamines on the action of (Mith)(2)-Co(II). Our study suggests a novel method for enhancing the anticancer activity of DNA-binding metalloantibiotics through polyamine depletion.

The Influence of the Degradation of Tetracycline by Free Radicals from Riboflavin-5'-Phosphate Photolysis on Microbial Viability.

Tetracycline (TC) is a broad-spectrum antibiotic compound. Wastewater with TC may have an adverse effect on ecosystems. Riboflavin-5'-phosphate (FMN or flavin mononucleotide) is a non-toxic product of the phosphorylation of vitamin B2 and is required for the proper functioning of the humans. FMN is sensitized to ultraviolet (UV) and blue light radiation, as evidenced by the generation of reactive oxygen species (ROS). This study inspects feasible applications of blue light on FMN so as to develop a valid way of degrading TC by FMN photolysis. We used the increased rate of bacterial survival as a practical indicator of antibiotic degradation. TC in the presence of FMN solution decomposed completely after 20 W/m2 of blue light irradiation (TCF treatment), and the degradation of TC (D-TCF) occurred after the photolytic process. After TCF treatment, colony-forming units (CFUs) of Escherichia coli (E. coli) were determined for the D-TCF solution. The CFU of E. coli preservation was 93.2% of the D-TCF solution (50 µg/mL of TC in the presence of 114 µg/mL of FMN solution treated with 20 W/m2 of blue light irradiation at 25 °C for 1 h) cultivation. The mass spectrum of D-TCF showed diagnostic ion signals at m/z 431.0 and 414.0 Da. The molecular formula of D-TCF was C21H22N2O8, and the exact mass was 430.44 g/mol. TC degradation by FMN photolysis can significantly decrease the antimicrobial ability of TC. The results expressed here regarding the influence of FMN photolysis on TC degradation offer an environmentally sound wastewater treatment method.

Studies of sequence-specific DNA binding, DNA cleavage, and topoisomerase I inhibition by the dimeric chromomycin A3 complexed with Fe(II).

Chromomycin A3 (Chro) has been evidenced to exhibit much higher binding affinity toward Fe(II) by forming a highly stable 2:1 drug/metal complex, compared to its structural analogue, mithramycin (Mith). Different properties of the [(Chro)2-Fe(II)] complex acting on DNA, such as sequence specificity, DNA cleavage, and topoisomerase I (TopI) inhibition were studied. Kinetic analyses of surface plasmon resonance showed that the affinity of the [(Chro)2-Fe(II)] complex upon binding to hairpin DNA duplexes containing various tetranucleotide sequences follows the order: GGCC > CGCG > CCGG approximately GCGC > AGCT > ACGT > TGCA > TCGA. According to circular dichroism (CD) studies, most hairpin DNA duplexes appeared to retain their B-type conformations in the presence of the [(Chro)2-Fe(II)] complex, except the duplex containing the GGCC sequence, which exhibited the features of both A- and B-type DNA. In DNA-cleavage assays, the [(Chro) 2-Fe(II)] complex was shown to cause single-stranded cleavage of plasmid DNA because of a Fenton-type reaction. DNA cleavage activity of the [(Chro) 2-Fe(II)] complex was increased at low pH. Moreover, the complex was capable of inhibiting TopI activity. The [(Chro)2-Fe(II)] complex exhibited higher cytotoxicity than the [(Mith) 2-Fe(II)] complex in several cancer cell lines, most likely owing to its more stable dimeric structure and higher DNA-binding affinity. Our results provide significant evidence that the [(Chro)2-Fe(II)] complex could be promising in terms of its biological applications in the future.

Blue light induced free radicals from riboflavin in degradation of crystal violet by microbial viability evaluation.

Crystal violet (CV) is applied in daily use mainly as a commercial dye and antimicrobial agent. Waste water containing CV may affect aquatic ecosystems. Riboflavin, also known as vitamin B2, is non-toxic and an essential vitamin required for the functions of the human body. Riboflavin is photosensitive to UV and visible light in terms of generating reactive oxygen species. This study investigated the potential application of blue light on riboflavin, so as to come up with an effective way of degrading CV during its treatment. Photosensitivity of CV leading to degradation in the presence of riboflavin was investigated by light intensity, exposure time, and irradiation dosage. The degradation of CV during riboflavin photolysis treatment was studied by a UV/vis spectrometry and chromatography. The effects of CV degradation on microbial viability are relevant when considering the influences on the ecosystem. This study proved that riboflavin photochemical treatment with blue light degrades CV dye by ROS formation. The riboflavin photolysis-treated CV solution appeared to be transparent during conformational transformations of the CV that was rearranged by free radical species generated from riboflavin photolysis. After riboflavin photolysis, colony-forming units (CFUs) were determined for each CV solution. CFU preservation was 85.2% for the CV dissolved riboflavin solution treated with blue light irradiation at 2.0mW/cm2 for 120min. Degradation of CV by riboflavin photochemical procedures can greatly reduce antimicrobial ability and serve as an environmental friendly waste water treatment method. Our results presented here concerning riboflavin photolysis in degradation of CV provide a novel technique, and a simple and safe practice for environmental decontamination processes.

Blue light induced free radicals from riboflavin on E. coli DNA damage.

The micronutrients in many cellular processes, riboflavin (vitamin B(2)), FMN, and FAD are photo-sensitive to UV and visible light to generate reactive oxygen species (ROS). The riboflavin photochemical treatment with UV light has been applied for the inactivation of microorganisms to serve as an effective and safe technology. Ultra-violet or high-intensity radiation is, however, considered as a highly risky practice. This study was working on the application of visible LED lights to riboflavin photochemical reactions to development an effective antimicrobial treatment. The photosensitization of bacterial genome with riboflavin was investigated in vitro and in vivo by light quality and irradiation dosage. The riboflavin photochemical treatment with blue LED light was proved to be able to inactivate E. coli by damaging nucleic acids with ROS generated. Riboflavin is capable of intercalating between the bases of bacterial DNA or RNA and absorbs lights in the visible regions. LED light illumination could be a more accessible and safe practice for riboflavin photochemical treatments to achieve hygienic requirements in vitro.

Spermine attenuates the action of the DNA intercalator, actinomycin D, on DNA binding and the inhibition of transcription and DNA replication.

The anticancer activity of DNA intercalators is related to their ability to intercalate into the DNA duplex with high affinity, thereby interfering with DNA replication and transcription. Polyamines (spermine in particular) are almost exclusively bound to nucleic acids and are involved in many cellular processes that require nucleic acids. Until now, the effects of polyamines on DNA intercalator activities have remained unclear because intercalation is the most important mechanism employed by DNA-binding drugs. Herein, using actinomycin D (ACTD) as a model, we have attempted to elucidate the effects of spermine on the action of ACTD, including its DNA-binding ability, RNA and DNA polymerase interference, and its role in the transcription and replication inhibition of ACTD within cells. We found that spermine interfered with the binding and stabilization of ACTD to DNA. The presence of increasing concentrations of spermine enhanced the transcriptional and replication activities of RNA and DNA polymerases, respectively, in vitro treated with ActD. Moreover, a decrease in intracellular polyamine concentrations stimulated by methylglyoxal-bis(guanylhydrazone) (MGBG) enhanced the ACTD-induced inhibition of c-myc transcription and DNA replication in several cancer cell lines. The results indicated that spermine attenuates ACTD binding to DNA and its inhibition of transcription and DNA replication both in vitro and within cells. Finally, a synergistic antiproliferative effect of MGBG and ACTD was observed in a cell viability assay. Our findings will be of significant relevance to future developments in combination with cancer therapy by enhancing the anticancer activity of DNA interactors through polyamine depletion.

The impact of spermine competition on the efficacy of DNA-binding Fe(II), Co(II), and Cu(II) complexes of dimeric chromomycin A(3).

Chromomycin (Chro) forms a 2:1 drug/metal complex through the chelation with Fe(II), Co(II), or Cu(II) ion. The effects of spermine on the interaction of Fe(II), Co(II), and Cu(II) complexes of dimeric Chro with DNA were studied. Circular dichroism (CD) measurements revealed that spermine strongly competed for the Fe(II) and Cu(II) cations in dimeric Chro-DNA complexes, and disrupted the structures of these complexes. However, the DNA-Co(II)(Chro)(2) complex showed extreme resistance to spermine-mediated competition for the Co(II) cation. According to surface plasmon resonance (SPR) experiments, a 6mM concentration of spermine completely abolished the DNA-binding activity of Fe(II)(Chro)(2) and Cu(II)(Chro)(2) and interfered with the associative binding of Co(II)(Chro)(2) complexes to DNA duplexes, but only slightly affected dissociation. In DNA integrity assays, lower concentrations of spermine (1 and 2mM) promoted DNA strand cleavage by Cu(II)(Chro)(2), whereas various concentrations of spermine protected plasmid DNA from damage caused by either Co(II)(Chro)(2) or Fe(II)(Chro)(2). Additionally, DNA condensation was observed in the reactions of DNA, spermine, and Fe(II)(Chro)(2). Despite the fact that Cu(II)(Chro)(2) and Fe(II)(Chro)(2) demonstrated lower DNA-binding activity than Co(II)(Chro)(2) in the absence of spermine, while Cu(II)(Chro)(2) and Fe(II)(Chro)(2) exhibited greater cytoxicity against HepG2 cells than Co(II)(Chro)(2), possibly due to competition of spermine for Fe(II) or Cu(II) in the dimeric Chro complex in the nucleus of the cancer cells. Our results should have significant relevance to future developments in metalloantibiotics for cancer therapy.