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Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.

Chan YS, Wong JH, Fang EF, Pan W, Ng TB - PLoS ONE (2012)

Bottom Line: However, drastic reduction of the activity occurred at temperatures above 65 °C.Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells.Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong.

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 IC(50) 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.

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Flow cytometry analysis.Flow cytometry of (A) Annexin V-PI staining, (B) JC-1 staining and (C) cell cycle analysis on MCF7 cells treated with different concentrations of brown kidney bean lectin (BKBL) for 24 hrs. WinMDI 2.9 was used for data analysis.
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pone-0038961-g008: Flow cytometry analysis.Flow cytometry of (A) Annexin V-PI staining, (B) JC-1 staining and (C) cell cycle analysis on MCF7 cells treated with different concentrations of brown kidney bean lectin (BKBL) for 24 hrs. WinMDI 2.9 was used for data analysis.

Mentions: Flow cytometry was used for further investigations of BKBL-induced anti-proliferative activity. In Annexin V-FITC and PI staining of BKBL-treated MCF7 cells (Fig. 8A), the majority of the cells at the lower left quadrant had gradually shifted to the right as BKBL concentration increased, indicated increased phosphatidyl serine (PS) externalization. These were the cells entering the early apoptosis phase. Besides, the proportion of cells at the upper right quadrant also increased with an escalation of BKBL concentration, indicating more cells entering the late apoptosis phase and dying. In JC-1 staining of BKBL-treated MCF7 cells (Fig. 8B), the increase in BKBL concentration caused the majority of the cells to shift from upper left quadrant toward the lower right quadrant, indicating more cells were experiencing mitochondrial depolarization and undergoing cell death. In cell cycle analysis (Fig. 8C), after treatment with low concentrations of BKBL (0.44 µM, 1.33 µM), there was a mild increase in percentage of MCF7 cells in G2 phase, while that in G0/G1 phase decreased. There was a mild cell cycle arrest in G2 phase in MCF7 cells at low BKBL concentrations. However, when BKBL concentration was further increased, the percentage of MCF7 cells with damaged DNA had greatly increased. This signified significant damage of MCF7 cells at high BKBL concentrations.


Isolation of a glucosamine binding leguminous lectin with mitogenic activity towards splenocytes and anti-proliferative activity towards tumor cells.

Chan YS, Wong JH, Fang EF, Pan W, Ng TB - PLoS ONE (2012)

Flow cytometry analysis.Flow cytometry of (A) Annexin V-PI staining, (B) JC-1 staining and (C) cell cycle analysis on MCF7 cells treated with different concentrations of brown kidney bean lectin (BKBL) for 24 hrs. WinMDI 2.9 was used for data analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375228&req=5

pone-0038961-g008: Flow cytometry analysis.Flow cytometry of (A) Annexin V-PI staining, (B) JC-1 staining and (C) cell cycle analysis on MCF7 cells treated with different concentrations of brown kidney bean lectin (BKBL) for 24 hrs. WinMDI 2.9 was used for data analysis.
Mentions: Flow cytometry was used for further investigations of BKBL-induced anti-proliferative activity. In Annexin V-FITC and PI staining of BKBL-treated MCF7 cells (Fig. 8A), the majority of the cells at the lower left quadrant had gradually shifted to the right as BKBL concentration increased, indicated increased phosphatidyl serine (PS) externalization. These were the cells entering the early apoptosis phase. Besides, the proportion of cells at the upper right quadrant also increased with an escalation of BKBL concentration, indicating more cells entering the late apoptosis phase and dying. In JC-1 staining of BKBL-treated MCF7 cells (Fig. 8B), the increase in BKBL concentration caused the majority of the cells to shift from upper left quadrant toward the lower right quadrant, indicating more cells were experiencing mitochondrial depolarization and undergoing cell death. In cell cycle analysis (Fig. 8C), after treatment with low concentrations of BKBL (0.44 µM, 1.33 µM), there was a mild increase in percentage of MCF7 cells in G2 phase, while that in G0/G1 phase decreased. There was a mild cell cycle arrest in G2 phase in MCF7 cells at low BKBL concentrations. However, when BKBL concentration was further increased, the percentage of MCF7 cells with damaged DNA had greatly increased. This signified significant damage of MCF7 cells at high BKBL concentrations.

Bottom Line: However, drastic reduction of the activity occurred at temperatures above 65 °C.Flow cytometry with Annexin V and propidum iodide staining indicated apoptosis of MCF7 cells.Western blotting revealed that the lectin-induced apoptosis involved ER stress and unfolded protein response.

View Article: PubMed Central - PubMed

Affiliation: School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong.

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 IC(50) 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.

Show MeSH
Related in: MedlinePlus