Abstract
Calpain-3 is an intracellular cysteine protease, belonging to Calpain superfamily and predominantly expressed in skeletal muscle. In human melanoma cell lines and biopsies, we previously identified two novel splicing variants (hMp78 and hMp84) of Calpain-3 gene (CAPN3), which have a significant lower expression in vertical growth phase melanomas and, even lower, in metastases, compared to benign nevi. In the present study, in order to investigate the pathophysiological role played by the longer Calpain-3 variant, hMp84, in melanoma cells, we over-expressed it in A375 and HT-144 cells. In A375 cells, the enforced expression of hMp84 induces p53 stabilization, and modulates the expression of a few p53- and oxidative stress-related genes. Consistently, hMp84 increases the intracellular production of ROS (Reactive Oxygen Species), which lead to oxidative modification of phospholipids (formation of F2-isoprostanes) and DNA damage. Such events culminate in an adverse cell fate, as indicated by the decrease of cell proliferation and by cell death. To a different extent, either the antioxidant N-acetyl-cysteine or the p53 inhibitor, Pifithrin-α, recover cell viability and decrease ROS formation. Similarly to A375 cells, hMp84 over-expression causes inhibition of cell proliferation, cell death, and increase of both ROS levels and F2-isoprostanes also in HT-144 cells. However, in these cells no p53 accumulation occurs. In both cell lines, no significant change of cell proliferation and cell damage is observed in cells over-expressing the mutant hMp84C42S devoid of its enzymatic activity, suggesting that the catalytic activity of hMp84 is required for its detrimental effects. Since a more aggressive phenotype is expected to benefit from down-regulation of mechanisms impairing cell growth and survival, we envisage that Calpain-3 down-regulation can be regarded as a novel mechanism contributing to melanoma progression.
Highlights
Calpains (Clan CA-C2, EC 3.4.22.17) constitute a superfamily of Ca2+-regulated, intracellular cysteine proteases, evolutionarily well-conserved from bacteria to mammals
In order to over-express the Calpain-3 variant hMp84 in human melanoma cells, preliminary experiments performed in a few cell lines were aimed to produce stablytransfected cells, through selection of neomycin-resistant clones; we failed to obtain stable p84 over-expressing cells, since they all died after 7–10 days of selection
Muscular Dystrophy type 2A (LGMD2A), an autosomal recessive disease characterized by progressive atrophy and weakness of the proximal limb muscles [2,24]
Summary
Calpains (Clan CA-C2, EC 3.4.22.17) constitute a superfamily of Ca2+-regulated, intracellular cysteine proteases, evolutionarily well-conserved from bacteria to mammals. On the basis of expression profile, 6 out of the 15 mammalian genes are considered to be tissuespecific, and defects of the corresponding calpains have been associated with tissue-specific pathological phenotypes. Among these calpains, particular emphasis has been placed on CAPN3 gene product, Calpain-3 (or p94), predominantly expressed in skeletal muscle. Particular emphasis has been placed on CAPN3 gene product, Calpain-3 (or p94), predominantly expressed in skeletal muscle It proves to be crucial for muscle cell homeostasis, as demonstrated in Limb-Girdle Muscular Dystrophy type 2A (LGMD2A, or calpainopathy), which is characterized by different CAPN3 point mutations and by muscle hypotrophy, hypoplasia and myonuclear apoptosis [2,3]
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