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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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Results of MTT assay on different cell lines.The cells were treated with brown kidney bean lectin for (A) 24 hrs and (B) 48 hrs. Brown kidney bean lectin exerted strong anti-proliferative activity on MCF7 and CNE1 cells, mild anti-proliferative activity on HepG2 and CNE2 cells and weak anti-proliferative activity on NP69 cells. Results represent mean±SD (n = 3).
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pone-0038961-g006: Results of MTT assay on different cell lines.The cells were treated with brown kidney bean lectin for (A) 24 hrs and (B) 48 hrs. Brown kidney bean lectin exerted strong anti-proliferative activity on MCF7 and CNE1 cells, mild anti-proliferative activity on HepG2 and CNE2 cells and weak anti-proliferative activity on NP69 cells. Results represent mean±SD (n = 3).

Mentions: BKBL exerted anti-proliferative effects on some tumor cell lines. Based on the MTT assay, after 24-hour treatment with BKBL (Fig. 6A), viability of CNE1 and MCF7 cells was greatly reduced, with an IC50 of 3.12 µM and 5.12 µM, respectively. The anti-proliferative activity toward HepG2 and CNE2 cells was comparatively weaker; the IC50 values were 32.85 µM and 40.12 µM, respectively. On the other hand, BKBL only slightly inhibited proliferation of normal NP69 cells. When the treatment with BKBL was lengthened to 48 hours (Fig. 6B), the anti-proliferative effects of BKBL were more pronounced. While the IC50 value toward MCF7 cells was slightly reduced to 4.80 µM, the anti-proliferative activity on HepG2 and CNE2 became much more conspicuous, with the IC50 values reduced to 8.45 µM and 6.64 µM, respectively. However, normal NP69 cells were more responsive to BKBL with an IC50 of 16.70 µM, yet the effect was still much weaker than that on the tumor cell lines.


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)

Results of MTT assay on different cell lines.The cells were treated with brown kidney bean lectin for (A) 24 hrs and (B) 48 hrs. Brown kidney bean lectin exerted strong anti-proliferative activity on MCF7 and CNE1 cells, mild anti-proliferative activity on HepG2 and CNE2 cells and weak anti-proliferative activity on NP69 cells. Results represent mean±SD (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038961-g006: Results of MTT assay on different cell lines.The cells were treated with brown kidney bean lectin for (A) 24 hrs and (B) 48 hrs. Brown kidney bean lectin exerted strong anti-proliferative activity on MCF7 and CNE1 cells, mild anti-proliferative activity on HepG2 and CNE2 cells and weak anti-proliferative activity on NP69 cells. Results represent mean±SD (n = 3).
Mentions: BKBL exerted anti-proliferative effects on some tumor cell lines. Based on the MTT assay, after 24-hour treatment with BKBL (Fig. 6A), viability of CNE1 and MCF7 cells was greatly reduced, with an IC50 of 3.12 µM and 5.12 µM, respectively. The anti-proliferative activity toward HepG2 and CNE2 cells was comparatively weaker; the IC50 values were 32.85 µM and 40.12 µM, respectively. On the other hand, BKBL only slightly inhibited proliferation of normal NP69 cells. When the treatment with BKBL was lengthened to 48 hours (Fig. 6B), the anti-proliferative effects of BKBL were more pronounced. While the IC50 value toward MCF7 cells was slightly reduced to 4.80 µM, the anti-proliferative activity on HepG2 and CNE2 became much more conspicuous, with the IC50 values reduced to 8.45 µM and 6.64 µM, respectively. However, normal NP69 cells were more responsive to BKBL with an IC50 of 16.70 µM, yet the effect was still much weaker than that on the tumor cell lines.

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