Isolation of a Glucosamine Binding Leguminous Lectin with Mitogenic Activity towards Splenocytes and Anti-Proliferative Activity towards Tumor Cells

Abstract

A dimeric 64-kDa glucosamine-specific lectin was purified from seeds of Phaseolus vulgaris cv. “brown kidney bean.” The simple 2-step purification protocol involved affinity chromatography on Affi-gel blue gel and gel filtration by FPLC on Superdex 75. The lectin was absorbed on Affi-gel blue gel and desorbed using 1M NaCl in the starting buffer. Gel filtration on Superdex 75 yielded a major absorbance peak that gave a single 32-kDa band in SDS-PAGE. Hemagglutinating activity was completely preserved when the ambient temperature was in the range of 20°C–60°C. However, drastic reduction of the activity occurred at temperatures above 65°C. Full hemagglutinating activity of the lectin was observed at an ambient pH of 3 to 12. About 50% activity remained at pH 0–2, and only residual activity was observed at pH 13–14. Hemagglutinating activity of the lectin was inhibited by glucosamine. The brown kidney bean lectin elicited maximum mitogenic activity toward murine splenocytes at 2.5 µM. The mitogenic activity was nearly completely eliminated in the presence of 250 mM glucosamine. The lectin also increased mRNA expression of the cytokines IL-2, TNF-α and IFN-γ. The lectin exhibited antiproliferative activity toward human breast cancer (MCF7) cells, hepatoma (HepG2) cells and nasopharyngeal carcinoma (CNE1 and CNE2) cells with IC50 of 5.12 µM, 32.85 µM, 3.12 µM and 40.12 µM respectively after treatment for 24 hours. Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells. Hoechst 33342 staining also indicated formation of apoptotic bodies in MCF7 cells after exposure to brown kidney bean lectin. Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

Effect of Soaking, Cooking and Germination on Chemical Constituents and Bioactive Compounds as well as their Cytotoxic Activities of Black Bean Extracts

“…Our results are in agreement with previous studies of Chan et al [47] that demonstrated the antiproliferative activity of some components of beans, different from phenolic compounds, on human breast cancer (MCF7), human hepatoma (Hep G2) and nasopharyngeal carcinoma (CNE1 and CNE2) cell line.…”

ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

“…Interestingly, O -glycan-binding lectins may also induce responses in lymphocytes [ 54 , 55 ]. The effect of lectins on adaptive immune cells is usually approached by analyzing the occurrence of lymphoproliferative responses [ 56 , 57 , 58 , 59 , 60 , 61 ], which we previously verified in ArtinM-stimulated murine spleen cells [ 24 ]. Isolated CD4 + and CD8 + T cells also proliferated under ArtinM stimulation in a concentration-dependent manner.…”

Hematoporphyrin Monomethyl Ether-Mediated Photodynamic Therapy Selectively Kills Sarcomas by Inducing Apoptosis

“…Three-dimensional culture was first applied to PDT; we found that apoptosis of OS cells could be induced in 2D and 3D cell culture environments and that apoptosis is the main form of cell death by HMME-PDT. Formation of apoptotic bodies and chromatin condensation are remarkable features in apoptosis and can be visualized by the fluorescence of Hoechst 33342 lableing [20]. Chromatin condensation and nuclear pyknosis were observed with Hoechst 33342 in HMME-PDT groups.…”