Cellular and molecular effects of protons: apoptosis induction and potential implications for cancer therapy.

Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.

ESR study of the non-enzymic scission of xyloglucan by an ascorbate-H2O2-copper system: the involvement of the hydroxyl radical and the degradation of ascorbate.

Xyloglucan is degraded by a mixture of copper(II), hydrogen peroxide and ascorbate. In the presence of ascorbate and/or hydrogen peroxide, copper(II) species were rapidly reduced to copper(I), which react with hydrogen peroxide. Spin-trapping experiments showed that hydroxyl radicals formed and attacked xyloglucan causing its degradation. The formation of a carbon-centred ascorbyl (C-ascorbyl) radical and its degradation with the formation of oxalate, was also caused by hydroxyl radicals. As a consequence, the features of the bis(oxalate) copper(II) complex clearly appeared in the frozen solution ESR spectra. The formation of carbon-centred radicals on the xyloglucan is the trigger for a series of possible molecular rearrangements which led to its oxidative scission.

Copper(II) binding modes in the prion octapeptide PHGGGWGQ: a spectroscopic and voltammetric study.

The N-terminal octapeptide repeat region of human prion protein (PrPc) is known to bind Cu(II). To investigate the binding modes of copper in PrPc, an octapeptide Ac-PHGGGWGQ-NH2 (1), which corresponds to an octa-repeat sequence, and a tetrapeptide Ac-HGGG-NH2 (2) have been synthesised. The copper(II) complexes formed with 1 and 2 have been studied by circular dichroism (CD) and electron spin resonance (ESR) spectroscopy. Both peptides form 1:1 complexes with Cu(II) at neutral and basic pH. CD, ESR and visible absorption spectra suggest a similar co-ordination sphere of the metal ion in both peptides, which at neutral pH consists of a square pyramidal geometry with three peptidic nitrogens and the imidazole nitrogen as donor atoms. Cyclic voltammetric measurements were used to confirm the geometrical features of these copper(II) complexes: the observation of negative redox potentials are in good agreement with the inferred geometry. All these results taken together suggest that peptide 1 provides a single metal binding site to which copper(II) binds strongly at neutral and basic pH and that the binding of the metal induces the formation of a stiffened structure in the HGGG peptide fragment.

Study of H2O2 interaction with copper(II) complexes with diamino-diamide type ligands, diastereoisomeric dipeptides, and tripeptides.

Copper(II) complexes with LL or LD dipeptides, tripeptides, L-aminoacylamides, or N,N'-bis(aminoacyl)alcanediamine were studied in their reaction with hydrogen peroxide by ESR spectroscopy. Shifts in the magnetic parameters g parallel and A parallel or differences in the quenching percentages of the ESR signal intensity, due to the formation of copper(I) species, suggested that the decomposition mechanism of H2O2 proceeds through the formation of a five-coordinated adduct and a subsequent electron transfer. This last process gave rise to a decomposition process which involved not only H2O2, but also the ligand coordinated to copper. It was surprising to find that, at the longest interaction time, this decomposition reaction always produced a similar copper(II) complex with g = 2.330 +/- 0.005 and A = 164 +/- 4 x 10(-4) cm-1 in spite of the different ligands. Voltammetric measurements confirmed what had been seen by ESR spectroscopy, and suggested that the decomposition mechanism did not involve the formation of copper(III) species. Furthermore, the only copper(II) complex with diastereoisomeric dipeptides, which was able to promote the copper oxidation, was that formed by LL- or LD- Ala-Trp, thus suggesting that d-pi interaction plays a favorable role in the oxidation process. The complexes which showed catalytic activity in the hydrogen peroxide decomposition were those obtained from LL- or LD- Ala-Ala or Ala-Leu, i.e., copper(II) complexes with dipeptides having aliphatic side chains. This fact strongly supports the hypothesis of the formation of a ligand radical species due to the breakage of the weak copper(I)-peptide nitrogen bond, radical starting the degradation of the ligand itself.

Copper(II) complexes encapsulated in human red blood cells.

Copper(II) complexes were encapsulated in human red blood cells in order to test their possible use as antioxidant drugs by virtue of their labile character. ESR spectroscopy was used to verify whether encapsulation in red blood cells leads to the modification of such complexes. With copper(II) complexes bound to dipeptides or tripeptides, an interaction with hemoglobin was found to be present, the hemoglobin having a strong coordinative site formed by four nitrogen donor atoms. Instead, with copper(II) complexes with TAD or PheANN3, which have the greatest stability. ESR spectra always showed the original species. Only the copper(II) complex with GHL gave rise to a complicated behavior, which contained signals from iron(III) species probably coming from oxidative processes. Encapsulation of all copper(II) complexes in erythrocytes caused a slight oxidative stress, compared to the unloaded and to the native cells. However, no significant differences were observed in the major metabolic properties (GSH, glycolytic rate, hexose monophosphate shunt, Ca(2+)-ATPase) of erythrocytes loaded with different copper(II) complexes, with the exception of methemoglobin levels, which were markedly increased in the case of [Cu(GHL)H-1] compared to [Cu(TAD)]. This latter finding suggests that methemoglobin formation can be affected by the type of complex used for encapsulation, depending on the direct interaction of the copper(II) complex with hemoglobin.