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Scarring is a significant medical burden; financially to the health care system and physically and psychologically for patients. Importantly, there have been numerous case reports describing the occurrence of cancer in burn scars. Currently available therapies are not satisfactory due to their undesirable side-effects, complex delivery routes, requirements for long-term use and/or expense. Radix Arnebiae (Zi Cao), a perennial herb, has been clinically applied to treat burns and manage scars for thousands of years in Asia. Shikonin, an active component extracted from Radix Arnebiae, has been demonstrated to induce apoptosis in cancer cells. Apoptosis is an essential process during scar tissue remodelling. It was therefore hypothesized that Shikonin may induce apoptosis in scar-associated cells. This investigation presents the first detailed in vitro study examining the functional responses of scar-associated cells to Shikonin, and investigates the mechanisms underlying these responses. The data obtained suggests that Shikonin inhibits cell viability and proliferation and reduces detectable collagen in scar-derived fibroblasts. Further investigation revealed that Shikonin induces apoptosis in scar fibroblasts by differentially regulating the expression of caspase 3, Bcl-2, phospho-Erk1/2 and phospho-p38. In addition, Shikonin down-regulates the expression of collagen I, collagen III and alpha-smooth muscle actin genes hence attenuating collagen synthesis in scar-derived fibroblasts. In summary, it is demonstrated that Shikonin induces apoptosis and decreases collagen production in scar-associated fibroblasts and may therefore hold potential as a novel scar remediation therapy.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Culture extracts from the cave-derived actinomycete Nonomuraea specus were investigated, resulting in the discovery of a new S-bridged pyronaphthoquinone dimer and its monomeric progenitors designated hypogeamicins A-D (1-4). The structures were elucidated using NMR spectroscopy, and the relative stereochemistries of the pyrans were inferred using NOE and comparison to previously reported compounds. Absolute stereochemistry was determined using quantum chemical calculations of specific rotation and vibrational and electronic circular dichroism spectra, after an extensive conformational search and including solute-solvent polarization effects, and comparing with the corresponding experimental data for the monomeric congeners. Interestingly, the dimeric hypogeamicin A (1) was found to be cytotoxic to the colon cancer derived cell line TCT-1 at low micromolar ranges, but not bacteria, whereas the monomeric precursors possessed antibiotic activity but no significant TCT-1 cytotoxicity.
Cells adapt to stressors by activating mechanisms that repair damage and protect them from further injury. Stress-induced damage accumulates with age and contributes to age associated diseases. Increased age attenuates the ability to mount a stress response, but little is known about the mechanisms by which this occurs. To begin addressing this problem, we studied hormesis in the nematode Caenorhabditis elegans. When exposed to a low concentration of the xenobiotic juglone, young worms mount a robust hormetic stress response and survive a subsequent exposure to a higher concentration of juglone that is normally lethal to naïve animals. Old worms are unable to mount this adaptive response. Microarray and RNAi analyses demonstrate that an altered transcriptional response to juglone is responsible in part for the reduced adaptation of old worms. Many genes differentially regulated in young versus old animals are known or postulated to be regulated by the FOXO homologue DAF-16 and the Nrf2 homologue SKN-1. Activation of these pathways is greatly reduced in juglone stressed old worms. DAF-16- and SKN-1-like transcription factors play highly conserved roles in regulating stress resistance and longevity genes. Our studies provide a foundation for developing a molecular understanding of how age affects cytoprotective transcriptional pathways.
Cytochrome P450 158A2 (CYP158A2) has been shown to catalyze an unusual oxidative C-C coupling reaction to polymerize flaviolin and form highly conjugated pigments (three isomers of biflaviolin and one triflaviolin) in Streptomyces coelicolor A3(2) which protect the soil bacterium from deleterious effects of UV irradiation (Zhao B. et al. (2005) J. Biol. Chem. 280, 11599-11607). The present studies demonstrate that the subfamily partner CYP158A1, sharing 61% amino acid identity with CYP158A2, can also catalyze the same flaviolin dimerization reactions, but it generates just two of the three isomers of biflaviolin that CYP158A2 produces. Furthermore, the two CYP158A1 products have very different molar ratios compared with the corresponding CYP158A2 products, indicating that each enzyme maintains its own stereo- and regiospecificity. To find an explanation for these differences, three CYP158A1 structures have been solved by X-ray crystallography and have been compared with those for CYP158A2. The structures reveal surprising differences. Particularly, only one flaviolin molecule is present close to the heme iron in CYP158A1, and the second flaviolin molecule binds at the entrance of the putative substrate access channel on the protein distal surface 9 A away. Our work describes two members of the same P450 subfamily, which produce the same products by oxidative C-C coupling yet show very different structural orientations of substrate molecules in the active site.
Cytochrome P450 158A2 (CYP158A2) can polymerize flaviolin to red-brown pigments, which may afford physical protection to the organism, possibly against the deleterious effects of UV radiation. We have found that the small molecule malonic acid enables cocrystallization of this mixed function oxidase with the azole inhibitor 4-phenylimidazole. The presence of malonate molecules affects the behavior of the binding of 4-phenylimidazole to CYP158A2 and increases inhibition potency up to 2-fold compared to 4-phenylimidazole alone. We report here the crystal structure of the 4-phenylimidazole/malonate complex of CYP158A2 at 1.5 A. Two molecules of malonate used in crystallization are found above the single inhibitor molecule in the active site. Those two molecules are linked between the BC loop and beta 1-4/beta 6-1 strands via hydrogen bond interactions to stabilize the conformational changes of the BC loop and beta strands that take place upon inhibitor binding compared to the ligand-free structure we have reported previously. 4-Phenylimidazole can launch an extensive hydrogen-bonding network in the region of the F/G helices which may stabilize the conformational changes. Our findings clearly show that two molecules of malonate assist the inhibitor 4-phenylimidazole to assume a specific location producing more inhibition in the enzyme catalytic activity.
Cytochrome P450 158A2 (CYP158A2) is encoded within a three-gene operon (sco1206-sco1208) in the prototypic soil bacterium Streptomyces coelicolor A3(2). This operon is widely conserved among streptomycetes. CYP158A2 has been suggested to produce polymers of flaviolin, a pigment that may protect microbes from UV radiation, in combination with the adjacent rppA gene, which encodes the type III polyketide synthase, 1,3,6,8-tetrahydroxynaphthalene synthase. Following cloning, expression, and purification of this cytochrome P450, we have shown that it can produce dimer and trimer products from the substrate flaviolin and that the structures of two of the dimeric products were established using mass spectrometry and multiple NMR methods. A comparison of the x-ray structures of ligand-free (1.75 angstroms) and flaviolin-bound (1.62 angstroms) forms of CYP158A2 demonstrates a major conformational change upon ligand binding that closes the entry into the active site, partly due to repositioning of the F and G helices. Particularly interesting is the presence of two molecules of flaviolin in the closed active site. The flaviolin molecules form a quasi-planar three-molecule stack including the heme of CYP158A2, suggesting that oxidative C-C coupling of these phenolic molecules leads to the production of flaviolin dimers.
Two new EPR approaches were developed for determination of rate constants of reaction glutathione (GSH), N-(2-mercaptopropionyl) glycine (MPG), dihydrolipoic acid (BNL), and tetranor-dihydrolipoic acid (TNL) with superoxide radical. In both cases the competition between thiols and spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) for superoxide radical was used. In the first method the dependence of amplitude of EPR spectrum of DMPO-OOH spin adduct on concentration of thiols in a superoxide-generating system was studied. In the second approach the changes in reduced thiol concentration due to reaction with superoxide radical were measured by nitroxide biradical containing disulfide bond. Observed rate constants were the following: GSH, 1.8 x 10(5) M-1s-1; MPG, 2.2 x 10(5) M-1s-1; TNL, 1.2 x 10(5) M-1s-1; BNL, 2.5 x 10(5) M-1s-1; DHL, 4.8 x 10(5) M-1s-1. The determination of the rate constants of reaction of superoxide radical with thiols by spectrophotometrical cytochrome C assay could result in an underestimation of the values due to the reduction of cytochrome C by thiols. Use of epinephrine for this purpose could lead to an overestimation of experimental rate constants because the adrenochrome formed in the reaction of epinephrine with superoxide radical reacts with thiols.
For the first time the covalent binding of anticancer anthracycline drugs and their potential synthetic analogs to oligonucleotides of different sequences is proposed for obtaining site-specific DNA scission in systems in vitro and in vivo. New compounds such as daunomycin (Dm) and synthetic naphthoquinone (NQ), covalently bound to the heptadeoxynucleotide of pCCAAACA (Dm-pN7) and decadeoxythymidilate (pT10p-NQ), have been obtained. These oligonucleotide derivatives can form specific complexes with complementary oligonucleotide sequences; these compounds and their complementary complexes can be reduced by purified NADPH-cytochrome P-450 reductase. Using the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), it has been shown that in aerobic conditions Dm-pN7 and pT10p-NQ are capable of generating OH radicals with and without complementary oligonucleotides. The chemical stability of the compounds in redox reactions has been studied. Oligonucleotide derivatives of natural and synthetic quinones capable of generating OH radicals seem to be a promising tool for site-specific scission of DNA in solution and in cells.