Molecular mechanism of the unusual biphasic effects of the natural compound hinokitiol on iron-induced cellular DNA damage.

Hinokitiol is a natural monoterpene compound found in the heartwood of cupressaceous plants that have anticancer and anti-inflammatory properties. However, few studies have focused on its effect on iron-mediated cellular DNA damage. Here we show that hinokitiol exhibited unusual biphasic effects on iron-induced DNA damage in a molar ratio (hinokitiol/iron) dependent manner in HeLa cells. Under low ratios (<3:1), hinokitiol markedly enhanced DNA damage induced by Fe(II) or Fe(II)-H2O2; However, when the ratios increased over 3:1, the DNA damage was progressively inhibited. We found that the total cytoplasmic and nuclear iron concentration increased as the ratios of hinokitiol/iron increased. However, the cellular level of labile iron pool (LIP) only increased at ratios lower than 3, and the ROS generation is consistent with LIP change. Hinokitiol was found to interact with iron to form lipophilic hinokitiol-iron complexes with different stoichiometry and redox-activity by complementary applications of various analytical methods. Taken together, we propose that the enhancement of iron-induced cellular DNA damage by hinokitiol at low ratios (<3:1) was due to formation of lipophilic and redox-active iron complexes which facilitated cellular iron uptake and •OH production, while the inhibition at ratios higher than 3 was due to formation of redox-inactive iron complexes. These new findings will help us to design more effective drugs for the prevention and treatment of a series of iron-related diseases via regulating the two critical physicochemical factors (lipophilicity and redox activity of iron complexes) by simple natural compounds with iron-chelating properties.

Mechanism of synergistic DNA damage induced by caffeic acid phenethyl ester (CAPE) and Cu(II): Competitive binding between CAPE and DNA with Cu(II)/Cu(I).

Caffeic acid phenethyl ester (CAPE) is an active polyphenol of propolis from honeybee hives, and exhibits antioxidant and interesting pharmacological activities. However, in this study, we found that in the presence of Cu(II), CAPE exhibited pro-oxidative rather than antioxidant effect: synergistic DNA damage was induced by the combination of CAPE and Cu(II) together as measured by strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, which is dependent on the molar ratio of CAPE:Cu(II). Production of Cu(I) and H2O2 from the redox reaction between CAPE and Cu(II), and subsequent OH formation was found to be responsible for the synergistic DNA damage. DNA sequencing investigations provided more direct evidence that CAPE/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Interestingly, we found there are competitive binding between CAPE and DNA with Cu(II)/Cu(I), which changed the redox activity of Cu(II)/Cu(I), via complementary applications of different analytical methods. The observed DNA damage was mainly attributed to the formation of DNA-Cu(II)/Cu(I) complexes, which is still redox active and initiated the redox reaction near the binding site between copper and DNA. Based on these data, we proposed that the synergistic DNA damage induced by CAPE/Cu(II) might be due to the competitive binding between CAPE and DNA with Cu, and site-specific production of OH near the binding site of copper with DNA. Our findings may have broad biological implications for future research on the pro-oxidative effects of phenolic compounds in the presence of transition metals.

Diethyldithiocarbamate-copper nanocomplex reinforces disulfiram chemotherapeutic efficacy through light-triggered nuclear targeting.

To circumvent the huge cost, long R&D time and the difficulty to identify the targets of new drugs, repurposing the ones that have been clinically approved has been considered as a viable strategy to treat different diseases. In the current study, we outlined the rationale for repurposing disulfiram (DSF, an old alcohol-aversion drug) to treat primary breast cancer and its metastases. Methods: To overcome a few shortcomings of the individual administration of DSF, such as the dependence on copper ions (Cu2+) and limited capability in selective targeting, we here artificially synthesized the active form of DSF, diethyldithiocarbamate (DTC)-Cu complex (CuET) for cancer therapeutics. To achieve a greater efficacy in vivo, smart nanomedicines were devised through a one-step self-assembly of three functional components including a chemically stable and biocompatible phase-change material (PCM), the robust anticancer drug (CuET) and a near-infrared (NIR) dye (DIR), namely CuET/DIR NPs. A number of in vitro assays were performed including the photothermal efficacy, light-triggered drug release behavior, nuclear localization, DNA damage and induction of apoptosis of CuET/DIR NPs and molecular mechanisms underlying CuET-induced repression on cancer metastatic behaviors. Meanwhile, the mice bearing 4T1-LG12-drived orthotopic tumors were employed to evaluate in vivo biodistribution and anti-tumor effect of CuET/DIR NPs. The intravenous injection model was employed to reflect the changes of the intrinsic metastatic propensity of 4T1-LG12 cells responding to CuET/DIR NPs. Results: The rationally designed nanomedicines have self-traceability for bioimaging, long blood circulation time for enhanced drug accumulation in the tumor site and photo-responsive release of the anticancer drugs. Moreover, our data unearthed that CuET/DIR nanomedicines behave like "Trojan horse" to transport CuET into the cytoplasm, realizing substantial intracellular accumulation. Upon NIR laser irradiation, massive CuET would be triggered to release from the nanomedicines and reach a high local concentration towards the nucleus, where the pro-apoptotic effects were conducted. Importantly, our CuET/DIR nanomedicines revealed a pronounced capability to leash breast cancer metastases through inhibition on EMT. Additionally, these nanomedicines showed great biocompatibility in animals. Conclusion: These combined data unearthed a remarkably enhanced tumor-killing efficacy of our CuET nanomedicines through nuclear targeting. This work may open a new research area of repurposing DSF as innovative therapeutic agents to treat breast cancer and its metastases.

Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn(III): First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location.

Isoniazid (INH), the most-widely used anti-tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon-centered isonicotinic acyl radical, which was considered to be responsible for its anti-tuberculosis activity. However, it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal- or proximal-nitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn(III) pyrophosphate. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.

Mechanism of synergistic DNA damage induced by the hydroquinone metabolite of brominated phenolic environmental pollutants and Cu(II): Formation of DNA-Cu complex and site-specific production of hydroxyl radicals.

2,6-Dibromohydroquinone (2,6-DBrHQ) has been identified as an reactive metabolite of many brominated phenolic environmental pollutants such as tetrabromobisphenol-A (TBBPA), bromoxynil and 2,4,6-tribromophenol, and was also found as one of disinfection byproducts in drinking water. In this study, we found that the combination of 2,6-DBrHQ and Cu(II) together could induce synergistic DNA damage as measured by double strand breakage in plasmid DNA and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) formation, while either of them alone has no effect. 2,6-DBrHQ/Cu(II)-induced DNA damage could be inhibited by the Cu(I)-specific chelating agent bathocuproine disulfonate and catalase, but not by superoxide dismutase, nor by the typical hydroxyl radical (•OH) scavengers such as DMSO and mannitol. Interestingly, we found that Cu(II)/Cu(I) could be combined with DNA to form DNA-Cu(II)/Cu(I) complex by complementary application of low temperature direct ESR, circular dichroism, cyclic voltammetry and oxygen consumption methods; and the highly reactive •OH were produced synergistically by DNA-bound-Cu(I) with H2O2 produced by the redox reactions between 2,6-DBrHQ and Cu(II), which then immediately attack DNA in a site-specific manner as demonstrated by both fluorescent method and by ESR spin-trapping studies. Further DNA sequencing investigations provided more direct evidence that 2,6-DBrHQ/Cu(II) caused preferential cleavage at guanine, thymine and cytosine residues. Based on these data, we proposed that the synergistic DNA damage induced by 2,6-DBrHQ/Cu(II) might be due to the synergistic and site-specific production of •OH near the binding site of copper and DNA. Our findings may have broad biological and environmental implications for future research on the carcinogenic polyhalogenated phenolic compounds.

Ascorbate does not act as a pro-oxidant towards lipids and proteins in human plasma exposed to redox-active transition metal ions and hydrogen peroxide.

The combination of ascorbate, transition metal ions, and hydrogen peroxide (H(2)O(2)) is an efficient hydroxyl radical generating system called "the Udenfriend system." Although the pro-oxidant role of ascorbate in this system has been well characterized in vitro, it is uncertain whether ascorbate also acts as a pro-oxidant under physiological conditions. To address this question, human plasma, used as a representative biological fluid, was either depleted of endogenous ascorbate with ascorbate oxidase, left untreated, or supplemented with 25 microM-1 mM ascorbate. Subsequently, the plasma samples were incubated at 37 degrees C with 50 microM-1 mM iron (from ferrous ammonium sulfate), 60 or 100 microM copper (from cupric sulfate), and/or 200 microM or 1 mM H(2)O(2). Although endogenous and added ascorbate was depleted rapidly in the presence of transition metal ions and H(2)O(2), no cholesterol ester hydroperoxides or malondialdehyde were formed, i.e., ascorbate protected against, rather than promoted, lipid peroxidation. Conversely, depletion of endogenous ascorbate was sufficient to cause lipid peroxidation, the rate and extent of which were enhanced by the addition of metal ions but not H(2)O(2). Ascorbate also did not enhance protein oxidation in plasma exposed to metal ions and H(2)O(2), as assessed by protein carbonyl formation and depletion of reduced thiols. Interestingly, neither the rate nor the extent of endogenous alpha-tocopherol oxidation in plasma was affected by any of the treatments. Our data show that even in the presence of redox-active iron or copper and H(2)O(2), ascorbate acts as an antioxidant that prevents lipid peroxidation and does not promote protein oxidation in human plasma in vitro.