A novel copper (II) complex activated both extrinsic and intrinsic apoptotic pathways in liver cancerous cells

Recent advances have put fundamental focus on the application of copper (II) (Cu [II]) complexes as agents for fighting against cancer. To determine whether [Cu(L)(2imi)] complex as a novel Cu complex can induce apoptosis in HepG2 as cancerous cells and L929 as normal cells via extrinsic or intrinsic apoptotic pathways, both cell lines were treated for 24 and 48 hours at IC₅₀ concentrations of [Cu(L)(2imi)] complex. Then, the expression of some apoptosis-related genes including p53, caspase-8, bcl-2, and bax were assayed by real-time polymerase chain reaction. The [Cu(L)(2imi)] complex seems to inhibit the expression of bcl-2 in complex-treated HepG2 cancerous cells following the 24- and 48-hour treatment. The complex upregulated the p53, bax, and caspase-8 genes, therefore treatment of HepG2 cancerous cells with [Cu(L)(2imi)] complex induces programmed cell death via the upregulation of relative bax/bcl-2 ratio. Finally, this copper complex triggered apoptosis in HepG2 cells via both intrinsic and extrinsic pathway, whereas treatment of normal L929 cells with this complex induce apoptosis only via intrinsic pathway with the upregulation of relative bax/bcl-2 ratio and does not affect the expression level of caspase-8 gene and does not trigger the extrinsic pathway. Finally, these results obtained from present study confirm the role of a novel Cu complex on the induction of apoptosis process in HepG2 and L929 cells by overexpression of bax, inhibition of bcl-2 and increase of the relative bax/bcl-2 ratio. These results support that the [Cu(L)(2imi)] complex is able to induce apoptosis in cancerous cells, therefore, it has a potential for development as a novel anticancer drug.     

Identification of sodC encoding periplasmic [Cu,Zn]-superoxide dismutase in Salmonella

Abstract sodC , encoding [Cu,Zn]-cofactored Superoxide dismutase, once thought to be virtually confined to eukaryotes, has now been described in many Gram-negative pathogens that have their primary niche of colonization in the upper respiratory tract. Their role in host-parasite interactive biology is unknown. We here show that members of the major human and animal enteric pathogenic species Salmonella harbour a version of sodC most closely resembling that found in Brucella abortus . The enzyme it encodes is a novel candidate determinant of virulence in Salmonella , an intracellular pathogen potentially exposed to toxic oxygen free radicals within its intracellular niche.     

Topical superoxide dismutase reduces post-irradiation breast cancer fibrosis

Fibrosis following breast radiotherapy for mammary cancer is a frequent undesired effect with objective (esthetic) and subjective (pain) consequences. Forty-four patients with clinical radiofibrosis following conservative treatment of breast cancer were evaluated for the local antifibrosis effect of copper zinc superoxide dismutase [SOD(Cu/Zn)]. Extracted SOD(Cu/Zn) in a concentration of 3,600 units/mg was applied as ointment to the fibrotic affected area, b.i.d. for 90 days, in a total dose of 40 mg. The radiofibrosis intensity was scored on the basis of clinical criteria (pain and the fibrosis area) before and after SOD(Cu/Zn) treatment. SOD(Cu/Zn) was found to be effective in reducing radiation induced fibrosis by a lowering pain score in 36/39 patients and a decrease of the fibrotic area size in half cases, after 6 months. The intensity and changes of breast fibrosis were assessed also by mammography and, for the topographical distribution of subcutaneous temperature, by infrared thermography. Mammography density suggested decreased fibrosis in one third of patients. Thermography showed that fibrosis was accompanied by two zones clinically indistinctive: a central area with maximum thermal activity, called "Maximal Thermic zone" (MTZ) and a peripheral area with less thermal activity but higher than in the surrounding normal tissue, "Transitional Thermic Zone" (TTZ). Both MTZ and TTZ were significantly decreased in 36/44 patients after SOD(Cu/Zn) treatment. Clinical changes persisted all along the study. Treatment was well tolerated except for one case of local allergic reaction, and no important side effects. Molecular mechanisms involved are discussed. Further studies are running to confirm and explain these results.     

Modification of cysteine 111 in Cu/Zn superoxide dismutase results in altered spectroscopic and biophysical properties

Cu/Zn superoxide dismutase (SOD) mutations are involved in about 20% of all cases of familial amyotrophic lateral sclerosis (FALS). Recently, it has been proposed that aberrant copper activity may be occurring within SOD at an alternative binding, and cysteine 111 has been identified as a potential copper ligand. Using a commercial source of human SOD isolated from erythrocytes, an anomalous absorbance at 325 nm was identified. This unusual property, which does not compromise SOD activity, had previously been shown to be consistent with a sulfhydryl modification at a cysteine residue. Here, we utilized limited trypsin proteolysis and mass spectrometry to show that the modification has a mass of 32 daltons and is located at cysteine 111. The reaction of SOD with sodium sulfide, which can react with cysteine to form a persulfide group, and with potassium cyanide, which can selectively remove persulfide bonds, confirmed the addition of a persulfide group at cysteine 111. Gel electrophoresis and glutaraldehyde cross-linking revealed that this modification makes the acid-induced denaturation of SOD fully irreversible. Furthermore, the modified protein exhibits a slower acid-induced unfolding, and is more resistant to oxidation-induced aggregation caused by copper and hydrogen peroxide. Thus, these results suggest that cysteine 111 can have a biochemical and biophysical impact on SOD, and suggest that it can interact with copper, potentially mediating the copper-induced oxidative damage of SOD. It will be of interest to study the role of cysteine 111 in the oxidative damage and aggregation of toxic SOD mutants.     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

Folding of Cu, Zn superoxide dismutase and familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.     

Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie-infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPc is transformed into PrPSc in prion pathologies. The complex cross-talk between PrPc and cholinergic neurons was analyzed in vitro using peroxynitrite and Cu2+ treatments on nerve endings isolated from Torpedo marmorata, a model of the motoneuron pre-synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration-dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+ (in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell-shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)-induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.     

Iron porphyrin treatment extends survival in a transgenic animal model of amyotrophic lateral sclerosis

Oxidative damage, produced by mutant Cu/Zn superoxide dismutase (SOD1), may play a role in the pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating motor neuron degenerative disease. A novel approach to antioxidant therapy is the use of metalloporphyrins that catalytically scavenge a wide range of reactive oxygen and reactive nitrogen species. In this study, we examined the therapeutic potential of iron porphyrin (FeTCPP) in the G93A mutant SOD1 transgenic mouse model of ALS. We found that intraperitoneal injection of FeTCPP significantly improved motor function and extended survival in G93A mice. Similar results were seen with a second group of mice wherein treatment with FeTCPP was initiated at the onset of hindlimb weakness-roughly equivalent to the time at which treatment would begin in human patients. FeTCPP-treated mice also showed a significant reduction in levels of malondialdehyde (a marker of lipid peroxidation), in total content of protein carbonyls (a marker of protein oxidation), and increased neuronal survival in the spinal cord. These results therefore provide further evidence of oxidative damage in a mouse model of ALS, and suggest that FeTCPP could be beneficial for the treatment of ALS patients.