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Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with beta-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed.

A thioredoxin reductase (TrxR) has been identified in the hyperthermophilic archaeon Sulfolobus solfataricus (Ss). This enzyme is a homodimeric flavoprotein that was previously identified as NADH oxidase in the same micro-organism ('Biotechnol. Appl. Biochem. 23 (1996) 47'). The primary structure of SsTrxR is made of 323 amino acid residues and contains two putative betaalphabeta regions for the binding of FAD, and a NADP(H) binding consensus sequence in the proximity of a CXXC motif. These findings indicate that SsTrxR is structurally related to the class II of the pyridine nucleotide-disulphide oxidoreductases family. Moreover, the enzyme exhibits a NADP(H) dependent thioredoxin reductase activity requiring the presence of FAD. Surprisingly, the reductase activity of SsTrxR is reduced in the presence of a specific inhibitor of mammalian TrxR. This finding demonstrates that the archaeal enzyme, although structurally related to eubacterial TrxR, is functionally closer to eukaryal enzymes. Experimental evidences indicate that a disulphide bridge is required for the reductase but also for the NADH oxidase activity of the enzyme. These results are further supported by the significantly reduced activities exerted by the C147A mutant. The integrity of the CXXC motif is also involved in the stability of the enzyme.