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Wedelactone (WL), a plant polyphenolic derivative of coumestan, represents a promising anti-cancer agent. The underlying mechanisms of its action are not fully understood and appear to involve interplay with copper ions. Herein, we examined coordination and redox interactions of WL with Cu 2+ in phosphate buffer (pH 7), and in two breast cancer cell lines. EPR, UV-Vis and fluorescence spectroscopy showed that WL and Cu 2+ build a coordination complex with 2 : 1 stoichiometry and distorted tetrahedral geometry. WL showed strong fluorescence that was quenched by Cu 2+ . The sequestration of the intracellular copper pool with neocuproine led to a significant drop in the cytotoxic effects of WL, whereas the co-application of Cu 2+ and WL and the formation of an extracellular complex suppressed both the cytotoxic effects of WL and copper loading. Fluorescence microscopy showed that WL is mainly localized in the cytosol and significantly less in the nuclei. WL fluorescence was stronger in cells pretreated with neocuproine, implying that the complex of WL and Cu 2+ is formed inside the cells. WL caused a two-fold increase in the lysosomal level of copper as well as copper-dependent lysosome membrane permeabilization. On the other hand, the protective effects of overexpression of thioredoxin 1 imply that WL exerts the main oxidative impact inside the nucleus. The interactions of WL with copper may be essential for therapeutic performance and selectivity against cancer cells, taking into account that a number of cancer types, including breast cancer, exhibit increased intratumoral copper levels or altered copper distribution.

Copper, zinc and sodium atomic line emissions in the plasma of a pencil discharge under the influence of easily ionizable elements (EIE), counterions and nitric acid were studied. These elements were introduced as the water solutions of nitrates and sulfates using an ICP-adopted nebulization system. The plasma pencil as a radiation source was operated in argon at 4 L min−1 powered by a 13.56 MHz generator at 140 W. The most intense depressing effect on the atomic emission of zinc (down to 15% of the unaffected intensity) and copper (down to 35%) appeared with sulfates. A lower effect was observed with chlorides (down to 70% for Zn and 30% for Cu) and the lowest effect was observed with nitrates, which increased the value for Zn by up to 40% and decreased that for Cu to about 50%. Nitric acid has a certain depression effect down to further 50% with salts, 80% for Cu, and 65% for Zn without salts; it also had a stabilization effect, partially compensating for the depression (double intensity for Cu with NaCl and HNO3 against sole NaCl) by anions and EIE. A combination of nitric acid with an added excess amount of an EIE salt could then provide predictable intensities. Sodium is a representative of EIE and it is a constituent of the introduced nitrate and sulfate salts. The sodium intensity dramatically decreased in the presence of sulfates, which might be a consequence of the reactions and energy exchange with the sulfur species in the plasma. [ABSTRACT FROM AUTHOR]

Wedelolactone is a multi-target natural plant coumestan exhibiting cytotoxicity towards cancer cells. Although several molecular targets of wedelolactone have been recognized, the molecular mechanism of its cytotoxicity has not yet been elucidated. In this study, we show that wedelolactone acts as an inhibitor of chymotrypsin-like, trypsin-like, and caspase-like activities of proteasome in breast cancer cells. The proteasome inhibitory effect of wedelolactone was documented by (i) reduced cleavage of fluorogenic proteasome substrates; (ii) accumulation of polyubiquitinated proteins and proteins with rapid turnover in tumor cells; and (iii) molecular docking of wedelolactone into the active sites of proteasome catalytic subunits. Inhibition of proteasome by wedelolactone was independent on its ability to induce reactive oxygen species production by redox cycling with copper ions, suggesting that wedelolactone acts as copper-independent proteasome inhibitor. We conclude that the cytotoxicity of wedelolactone to breast cancer cells is partially mediated by targeting proteasomal protein degradation pathway. Understanding the structural basis for inhibitory mode of wedelolactone might help to open up new avenues for design of novel compounds efficiently inhibiting cancer cells. [ABSTRACT FROM AUTHOR]

The influence of Easily Ionizable Elements (EIE) (chlorides of Na, K, Mg, Ca, Sr and Ba) on the measured spectral lines intensities of copper and zinc excited in a capacitively coupled tubular discharge, known as a plasma pencil, is described. A strong decrease was observed in the dependence on the concentration of the EIE. The decrease is not linear. Addition of NaCl, KCl or BaCl2 causes a steep decrease of the copper or zinc signal for a relatively narrow range of 0.05–0.80 g l−1. Other EIE, with relatively higher ionization energies, even caused an increase of the Cu I 324.754, 327.396, 510.554 and 578.213 nm and Zn I 213.856, 468.014, 472.216 and 481.053 nm signals. Higher EIE concentrations, up to 4 g l−1, bring only moderate signal suppression or even a weak signal increase. Atoms of Mg, Ca and Sr likely undergo preferential partial ionization but less than Na, K and Ba so that Cu and Zn atoms do not have enough energy for their ionization but still have enough energy for excitation. More Cu and Zn excited atoms, instead of ions, are then available and stronger Cu and Zn atomic emission is measured. Atoms of Na, K and Ba are so much ionized that Cu and Zn atoms even loose energy for their excitation. A stronger decrease was found for the copper line than for the zinc line. An explanation can be done through inelastic collisions between Cu and the EIE excited atoms with very similar energetic levels. The influence of the EIE on the plasma properties was also monitored by calculation of excitation (2900 ± 280) K, rotational temperatures (1000 ± 160) K and electron number density of (4.6 ± 0.7) × 1019 m−3; but no significant change was observable. [ABSTRACT FROM AUTHOR]