Abstract
Parasporin-2, a new crystal protein derived from noninsecticidal and nonhemolytic Bacillus thuringiensis, recognizes and kills human liver and colon cancer cells as well as some classes of human cultured cells. Here we report that a potent proteinase K-resistant parasporin-2 toxin shows specific binding to and a variety of cytocidal effects against human hepatocyte cancer cells. Cleavage of the N-terminal region of parasporin-2 was essential for the toxin activity, whereas C-terminal digestion was required for rapid cell injury. Protease-activated parasporin-2 induced remarkable morphological alterations, cell blebbing, cytoskeletal alterations, and mitochondrial and endoplasmic reticulum fragmentation. The plasma membrane permeability was increased immediately after the toxin treatment and most of the cytoplasmic proteins leaked from the cells, whereas mitochondrial and endoplasmic reticulum proteins remained in the intoxicated cells. Parasporin-2 selectively bound to cancer cells in slices of liver tumor tissues and susceptible human cultured cells and became localized in the plasma membrane until the cells were damaged. Thus, parasporin-2 acts as a cytolysin that permeabilizes the plasma membrane with target cell specificity and subsequently induces cell decay.
Highlights
The crystal (Cry)3 proteins produced in Bacillus thuringiensis, a Gram-positive bacterium, are known to show high cytotoxicity against insects [1]
Through our present analyses of its proteolytic activation and cytocidal effects, we show the following: (i) that parasporin-2 is highly activated through processing of both its N- and C-terminal propeptides; (ii) that it binds to the plasma membrane of hepatocyte cancer cells; (iii) that it rapidly increases the membrane permeability; and (iv) that it dramatically alters the cytoskeleton and organelle morphologies
After washing with phosphate-buffered saline (PBS), parasporin-2 was added to the cells in Dulbecco’s modified essential medium (DMEM) without fetal calf serum (FCS), and the cells were incubated for appropriate times
Summary
Cell Culture and Materials—HepG2 and COS-7 cells were cultured in Dulbecco’s modified essential medium (DMEM; Nissui) containing 10% fetal calf serum (FCS; Biological Industries), whereas HeLa cells were cultured in minimal essential medium (Nissui) containing 10% FCS, under 5% CO2 at 37 °C. For PI (Sigma) staining, cells (2 ϫ 104 cells/well) were grown on 96-well plates overnight and washed twice with PBS, before PI (final concentration: 5 mg/ml) in DMEM was added together with parasporin-2. After washing with PBS, parasporin-2 was added to the cells in DMEM without FCS, and the cells were incubated for appropriate times. The intoxicated, fixed, and permeabilized cells were treated with an anti-parasporin-2 antibody as the primary antibody, and labeled with Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes) as a fluorescent dye-conjugated secondary antibody, in PBS containing 10 mM glycine and 10% bovine serum albumin. After washing with PBS, secondary antibody/peroxidase-linked polymers were applied, and the sections were incubated with 100 ml of Tris-HCl, pH 7.6, containing 20 mg of 3,3Ј-diaminobenzidine tetrahydrochloride, 65 mg of sodium azide, and 20 ml of 30% H2O2. The diagnosis of each cancer tissue specimen was re-evaluated and confirmed by three pathologists who examined formalin-fixed and paraffin-embedded tissue sections stained with hematoxylin and eosin or appropriate immunohistochemical stains
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