calpain and Fibrosarcoma

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein which mediates staurosporine (STS) - induced cell death. AIF cleavage and translocation to the cytosol is thought to be calpain-1-dependent as calpain inhibitors reduce AIF proteolysis; however, many calpain inhibitors also inhibit matrix metalloproteinase-2 (MMP-2) activity, an intracellular and extracellular protease implicated in apoptosis. Here we investigated whether MMP-2 activity is affected in response to STS and if it contributes to AIF cleavage. Human fibrosarcoma HT1080 cells were treated with STS (0.1 µM, 0.25-24 h). A significant increase in cellular MMP-2 activity was seen by gelatin zymography after a 6 h STS treatment, prior to induction of cell necrosis. Western blot showed the time-dependent appearance of two forms of AIF (∼60 and 45 kDa) in the cytosol which were significantly increased at 6 h. Surprisingly, knocking down MMP-2 or inhibiting its activity with MMP-2 preferring inhibitors ARP-100 or ONO-4817, or inhibiting calpain activity with ALLM or PD150606, did not prevent the STS-induced increase in cytosolic AIF. These results show that although STS rapidly increases MMP-2 activity, the cytosolic release of AIF may be independent of the proteolytic activities of MMP-2 or calpain.

Topics: Apoptosis; Apoptosis Inducing Factor; Calpain; Cytosol; Fibrosarcoma; Humans; Matrix Metalloproteinase 2; Proteolysis; Staurosporine; Tumor Cells, Cultured

The pro-inflammatory cytokine Interleukin-1α (IL-1α) has recently emerged as a susceptibility marker for a wide array of inflammatory diseases associated with oxidative stress including Alzheimer's, arthritis, atherosclerosis, diabetes and cancer. In the present study, we establish that expression and nuclear localization of IL-1α are redox-dependent. Shifts in steady-state H2O2 concentrations (SS-[H2O2]) resulting from enforced expression of manganese superoxide dismutase (SOD2) drive IL-1α mRNA and protein expression. The redox-dependent expression of IL-1α is accompanied by its increased nuclear localization. Both IL-1α expression and its nuclear residency are abrogated by catalase co-expression. Sub-lethal doses of H2O2 also cause IL-1α nuclear localization. Mutagenesis revealed IL-1α nuclear localization does not involve oxidation of cysteines within its N terminal domain. Inhibition of the processing enzyme calpain prevents IL-1α nuclear localization even in the presence of H2O2. H2O2 treatment caused extracellular Ca(2+) influx suggesting oxidants may influence calpain activity indirectly through extracellular Ca(2+) mobilization. Functionally, as a result of its nuclear activity, IL-1α overexpression promotes NF-kB activity, but also interacts with the histone acetyl transferase (HAT) p300. Together, these findings demonstrate a mechanism by which oxidants impact inflammation through IL-1α and suggest that antioxidant-based therapies may prove useful in limiting inflammatory disease progression.

Topics: Calcium; Calpain; Cell Line, Tumor; Cell Nucleus; Cysteine; E1A-Associated p300 Protein; Fibrosarcoma; HEK293 Cells; Humans; Hydrogen Peroxide; Interleukin-1alpha; NF-kappa B; Oxidation-Reduction; Superoxide Dismutase

As part of a continuing search for potential anticancer drug candidates from antimicrobial peptides of marine organisms, tilapia (Oreochromis mossambicus) hepcidin TH2-3 was evaluated in several tumor cell lines. The results indicated that TH2-3, a synthetic 20-mer antimicrobial peptide, specifically inhibited human fibrosarcoma cell (HT1080 cell line) proliferation and migration. The way in which TH2-3 inhibited HT1080 cell growth was then studied. TH2-3 inhibited HT1080 cell growth in a concentration-dependent manner according to an MTT analysis, which was confirmed by a soft-agar assay and AO/EtBr staining. Scanning electron microscopy revealed that TH2-3 caused lethal membrane disruption in HT1080 cancer cells, and a wound healing assay supported that TH2-3 decreased the migration of HT1080 cells. In addition, c-Jun mRNA expression was downregulated after treatment with TH2-3 for 48-96 h compared to the untreated group. These findings suggest a mechanism of cytotoxic action of TH2-3 and indicate that TH2-3 may be a promising chemotherapeutic agent against human fibrosarcoma cells.

Topics: Animals; Antimicrobial Cationic Peptides; Antineoplastic Agents; Calpain; Caspase 3; Cathepsin G; Cathepsins; Cell Line; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Proliferation; Cell Survival; Chlorocebus aethiops; COS Cells; Down-Regulation; Fibrosarcoma; Fish Proteins; Gene Expression; HeLa Cells; Hepcidins; Humans; Proto-Oncogene Proteins c-jun; Serine Endopeptidases; Tilapia; Wound Healing

Cancer cells can invade three-dimensional matrices by distinct mechanisms, recently defined by their dependence on extracellular proteases, including matrix metalloproteinases. Upon treatment with protease inhibitors, some tumour cells undergo a 'mesenchymal to amoeboid' transition that allows invasion in the absence of pericellular proteolysis and matrix degradation. We show here that in HT1080 cells, this transition is associated with weakened integrin-dependent adhesion, consistently reduced cell surface expression of the alpha2beta1 integrin collagen receptor and impaired signalling downstream, as judged by reduced autophosphorylation of focal adhesion kinase (FAK). On examining cancer cells that use defined invasion strategies, we show that distinct from mesenchymal invasion, amoeboid invasion is independent of intracellular calpain 2 proteolytic activity that is usually needed for turnover of integrin-linked adhesions during two-dimensional planar migration. Moreover, an inhibitor of Rho/ROCK signalling, which specifically impairs amoeboid-like invasion, restores cell surface expression of alpha2beta1 integrin, downstream FAK autophosphorylation and calpain 2 sensitivity--features of mesenchymal invasion. These findings link weakened integrin function to a lack of requirement for calpain 2-mediated integrin adhesion turnover during amoeboid invasion. In keeping with the need for integrin adhesion turnover, mesenchymal invasion is uniquely sensitive to Src inhibitors. Thus, the need for a major pathway that controls integrin adhesion turnover defines and distinguishes cancer cell invasion strategies.

Topics: Base Sequence; Calpain; Carcinoma, Non-Small-Cell Lung; Cell Adhesion; Cell Line, Tumor; Fibrosarcoma; Flow Cytometry; Humans; Integrins; Lung Neoplasms; Mesoderm; Mutation, Missense; Neoplasm Invasiveness; RNA, Messenger; RNA, Small Interfering; src-Family Kinases

Purified plasma fibronectin in Tris-buffered saline aggregated on incubation at 37 degrees C in the presence of dithiothreitol without the presence of cells. On SDS polyacrylamide gel electrophoresis without reduction, dimeric fibronectin showed a 460 kDa band, while the protein band of aggregated fibronectin remained at the top of the running gel. The aggregate was a disulfide-bonded multimer, since both the dimeric and the multimeric fibronectins migrated as 230 kDa polypeptides after reduction. The multimer formation required SH reagent and proceeded faster with higher SH concentration, suggesting the occurrence of a disulfide exchange reaction during the aggregation. Since dimeric fibronectin with carboxymethylated sulfhydryl groups also formed multimers under the same condition, the free sulfhydryl groups of dimeric fibronectin may not be involved in the multimer formation, suggesting involvement of disulfide exchange from intramolecular bonds to intermolecular bonds. The multimerization was not influenced by Na+, Ca2+, or EDTA, while urea-treated fibronectin required a higher concentration of dithiothreitol for multimer formation. Fibronectin partially degraded by m-calpain did not form multimers. The multimeric fibronectin retained heparin-binding and cell attachment activities, but had lost gelatin-binding activity. Involvement of the terminal regions containing type I and type II repeats was suggested in the interaction of pFN leading to the multimerization.

Topics: Animals; Calpain; Cattle; Cell Adhesion; Cell-Free System; Chromatography, High Pressure Liquid; Disulfides; Dithiothreitol; Electrophoresis, Polyacrylamide Gel; Extracellular Matrix; Fibronectins; Fibrosarcoma; Molecular Weight; Polymers; Protein Binding; Tumor Cells, Cultured; Urea