title: Calpain-3 impairs cell proliferation and stimulates oxidative stress-mediated cell death in melanoma cells.
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.
authors: Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; Istituto Toscano Tumori (ITT), Firenze, Italy.
authors: Daniele
authors: D
authors: Moretti
authors: Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; Istituto Toscano Tumori (ITT), Firenze, Italy.
authors: Barbara
authors: B
authors: Del Bello
authors: Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; Istituto Toscano Tumori (ITT), Firenze, Italy.
authors: Giulia
authors: G
authors: Allavena
authors: Department of Translational Research and New Technologies in Medicine and Surgery, Medical School, University of Pisa, Pisa, Italy; Istituto Toscano Tumori (ITT), Firenze, Italy.
authors: Alessandro
authors: A
authors: Corti
authors: Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy.
authors: Cinzia
authors: C
authors: Signorini
authors: Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy; Istituto Toscano Tumori (ITT), Firenze, Italy.
authors: Emilia
authors: E
authors: Maellaro
mesh_terms: D052160:Benzothiazoles / Q000494:pharmacology; D002154:Calpain / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D006801:Humans; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D009124:Muscle Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D017382:Reactive Oxygen Species / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D052160:Benzothiazoles; D009124:Muscle Proteins; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D014050:Toluene; C121565:pifithrin; C105884:CAPN3 protein, human; D002154:Calpain