Growth inhibitory activity of a novel lectin from Cliona varians against K562 human erythroleukemia cells

Purpose in this study, the antitumoral potential of a novel lectin (CvL) purified from the marine sponge Cliona varians was studied in different cancer cell lines.Methods CvL cytotoxicity was evaluated in mammalian tumor cells and in normal human peripheral blood lymphocytes by the MTT assay using the same range of concentrations (1 - 150 mu g ml(-1)). the mechanisms involved in K562 cell death were investigated by confocal fluorescence microscopy, flow cytometry and immunoblot.Results CvL inhibited the growth of human leukemia cells, with IC(50) values of 70 and 100 mu g ml(-1) for K562 and JURKAT cells, respectively, but it was ineffective on blood lymphocytes and solid tumor cell lines. K562 cell death occurred 72 h after exposure to the lectin and with signs of apoptosis, as analyzed by DAPI and annexin V/PI staining. Investigation of the possible mediators of this process showed that cell death occurred via a caspase-independent pathway. Confocal fluorescence microscopy indicated a pivotal role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) abolished CvL cytotoxic eVect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B ( NF kappa B) expression in CvL-treated cells. These effects were accompanied by increased levels of p21 and reduced expression of pRb, suggesting that CvL can induce cell cycle arrest.Conclusions Collectively, these findings indicate an anti-leukemic eVect for CvL and suggest that cathepsin B acts as a death mediator in CvL-induced cytotoxicity possibly in an uncharacterized connection with the membrane death receptor pathway.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Financiadora de Estudos e Projetos (FINEP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv Fed Rio Grande do Norte, Dept Biofis & Farmacol, Ctr Biociencias, BR-59072970 Natal, RN, BrazilUniv Estadual Campinas, Dept Bioquim, Inst Biol, BR-13083970 Campinas, SP, BrazilUniv Fed Rio Grande do Norte, Dept Bioquim, Lab Quim & Funcao Proteinas Bioativas, BR-59072970 Natal, RN, BrazilUniv Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-08701970 Mogi Das Cruzes, SP, BrazilUniv Fed Rio Grande do Norte, Dept Bioquim, Ctr Biociencias, BR-59072970 Natal, RN, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, BrazilFAPESP: 06/07315-3Web of Scienc