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 μg 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 IC50 values of 70 and 100 μg 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 effect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NFκB) 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. 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