Laccase purified from Cerrena unicolor exerts antitumor activity against leukemic cells

Abstract

Chronic lymphocytic leukemia (CLL) is the most commonly observed adult hematological malignancy in Western countries. Despite the fact that recent improvements in CLL treatment have led to an increased percentage of complete remissions, CLL remains an incurable disease. Cerrena unicolor is a novel fungal source of highly active extracellular laccase (ex-LAC) that is currently used in industry. However, to the best of our knowledge, no reports regarding its anti-leukemic activity have been published thus far. In the present study, it was hypothesized that C. unicolor ex-LAC may possess cytotoxic activity against leukemic cell lines and CLL primary cells. C. unicolor ex-LAC was separated using anion exchange chromatography on diethylaminoethyl cellulose-Sepharose and Sephadex G-50 columns. The cytotoxic effects of ex-LAC upon 24- and 48-h treatment on HL-60, Jurkat, RPMI 8226 and K562 cell lines, as well as CLL primary cells of nine patients with CLL, were evaluated using 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay. Annexin V/propidium iodide staining of Jurkat cells treated with ex-LAC was used to investigate apoptosis via flow cytometry. Ex-LAC induced changes in Jurkat and RPMI 8226 cells, as visualized by fluorescence and scanning electron microscopy (SEM). The XTT assay revealed high cytotoxic rates following treatment with various concentrations of ex-LAC on all the cell lines and CLL primary cells analyzed, with a half maximal inhibitory concentration ranging from 0.4 to 1.1 µg/ml. Fluorescence microscopy and SEM observations additionally revealed apoptotic changes in Jurkat and RPMI 8226 cells treated with ex-LAC, compared with control cells. These results were in agreement with the apoptosis analysis of Jurkat cells on flow cytometry. In conclusion, C. unicolor ex-LAC was able to significantly induce cell apoptosis, and may represent a novel therapeutic agent for the treatment of various hematological neoplasms.

Anticancer, antioxidant, and antibacterial activities of low molecular weight bioactive subfractions isolated from cultures of wood degrading fungus Cerrena unicolor

“…The supernatant was immediately subdivided into two fractions on the ultrafiltration system Pellicon 2 Mini holder (Millipore, Bedford, MA, USA) with an Ultracel mini cartridge (10 kD cut-off). The starting fraction containing compounds with a molecular weight below 10 kDa had already been used as a source of low molecular weight metabolites (extracellular low molecular weight subfraction, ex-LMS) and had been tested as described in our previous publication [ 32 ]. The antioxidant and pro-oxidative properties, the antibacterial, antiviral, immunomodulatory, and anticancer activities, and the toxicity of the ex-LMS fraction have also been presented in previously published reports [ 30 – 33 ].…”

“…unicolor species possess cytotoxic and antiproliferative activity against cervical cancer cells (SiHa and CaSki) and melanomic cells [ 30 – 31 ]. The cytotoxic activity of laccase preparations towards several hematological malignancies has been described as well [ 32 ]. C .…”

Efficient propagation of suspended HL-60 cells in a disposable bioreactor supporting wave-induced agitation at various Reynolds number

“…Nonadherent hematopoietic cells are frequently recognized as ex vivo sources of protein-based bioproducts applied in biotechnology, and biomedicine as well. Up to now, HL-60 cells as valuable human cells, have been applied as model in many studies on blood cells differentiation and physiology [14][15][16][17], or as a simple in vitro model in determination of cytotoxicity of anti-leukemic drugs [18,19]. But the efficiency of in vitro cultures of suspended biomass of HL-60 cells has not been extensively discussed so far, and the growth of HL-60 cells maintained in disposable bioreactor, as well as the influence of hydrodynamic conditions of the wave-type agitation on proliferation of HL-60 cells, has not been deeply studied or analysed up to date.…”

“…However, hematopoietic cells are an acknowledged ex vivo source of bioproducts with many potential applications in bone marrow transplantation, immunotherapy, gene therapy, as well as the source of blood-derived products [13]. A HL-60 cell line, i.e., human acute promyelocytic leukemia cells, has been frequently applied as model of hematopoietic cells, and it was used in studies focused on in vitro blood cell formation [14], their physiology [15] and differentiation [16,17], as well as to studying the cytotoxicity effects of potential anti-leukemic drugs [18], or even in lab-on-a-chip applications [19]. Thus, an enhancement of HL-60 cells' propagation efficiency is a current research issue highly expected by representatives of a range of application niches, which experimentally involve such type of cells.…”

Effect of Ferulic Acid, a Phenolic Inducer of Fungal Laccase, on 26S Proteasome Activities In Vitro

“…Moreover, previous studies from our laboratory have shown that the ubiquitin-proteasome pathway is involved in the regulation of ligninolytic enzymes of white-rot fungi (such as laccase, benzenediol:oxygen oxidoreductase, EC 1.10.3.2) upon nutrient starvation [34][35][36] and in response to Cd 2+ exposure [33] and ER stress [37]. Lignin-degrading Basidiomycetes are increasingly being investigated due to their ecological significance and various potential applications of ligninolytic enzymes [38,39], especially laccase [40,41], in several industrial and biotechnological processes, including the application in the pulp and paper industry [42], enzymatic bioremediation [43], biosensor [44] and biofuel cell [45] construction, and medicine [46,47]. Numerous studies indicated that laccase production can be considerably stimulated by the presence of inducers, mainly aromatic or phenolic compounds related to lignin or lignin derivatives, such as ferulic acid, 2,5-xylidine, p-anisidine, or veratryl alcohol [48][49][50][51].…”