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. 2013 Oct;20(10):1159-69.
doi: 10.2174/0929866511320100011.

A Hemagglutinin From Northeast Red Beans With Immunomodulatory Activity and Anti-Proliferative and Apoptosis-Inducing Activities Toward Tumor Cells

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A Hemagglutinin From Northeast Red Beans With Immunomodulatory Activity and Anti-Proliferative and Apoptosis-Inducing Activities Toward Tumor Cells

Yau Sang Chan et al. Protein Pept Lett. .
Free PMC article

Abstract

A 64-kDa hemagglutinin from a Phaseolus vulgaris cultivar, the northeast red bean, was purified by a protocol composed of three chromatographic steps involving affinity chromatography on Affi-gel blue gel, cation exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The purified hemagglutinin appeared as a single 32-kDa band in SDS-PAGE indicating its dimeric nature. The N-terminal amino acid sequence of the hemagglutinin resembled the sequences of lectins and hemagglutinins from a number of Phaseolus species. The hemagglutinin manifested moderate thermostability and pH stability. It retained full activity up to 65 °C and in the pH range 2-12. It did not interact with simple sugars such as glucose, mannose and galactose. The hemagglutinin exerted immunostimulatory effects by upregulating the expression of cytokines like interferon-γ and tumor necrosis factor-α. It also exhibited antiproliferative activity on a number of tumor cells including MCF7 (breast cancer), HepG2 (liver cancer), CNE1 and CNE2 (nasopharyngeal cancer) cells, with stronger activity toward MCF7 and CNE1 cells. The hemagglutinin induced phophatidylserine externalization, mitochondrial depolarization and DNA condensation in MCF7 cells, indicating initiation of apoptosis. However, at high hemagglutinin concentrations, severe damage to the MCF7 cells was detected.

Conflict of interest statement

The authors confirm that this article content has no conflict of interest.

Figures

Figure 1
Figure 1
Elution profile of northeast red bean hemagglutinin from a Superdex 75 HR 10/300 gel filtration column. The shaded region contained purified hemagglutinin.
Figure 2
Figure 2
SDS-PAGE for purification of northeast red bean hemagglutinin. Lane 1: crude extract of northeast red beans. Lane 2: bound fraction from Affi-gel blue gel. Lane 3: unbound fraction from SP-Sepharose. Lane 4: Purified lectin from Superdex 75. M: protein markers from GE Healthcare including phosphorylase b (97 kDa), bovine serum albumin (67 kDa), carhonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa), and α- lactoalbumin (14.4 kDa).
Figure 3
Figure 3
Induction of gene expression of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) of mouse splenocytes by northeast red bean hemagglutinin and concanavalin A. (GAPDH = glyceraldehydes-3-phosphate dehydrogenase, IFN-γ = interferon-γ, and TNF-α = tumor necrosis factor-α)
Figure 4
Figure 4
Results of MTT assay on inhibition of CNE1, MCF7, HepG2 and CNE2 cells after treatment with northeast red bean hemagglutinin for 24 hrs. Results represent mean±SD (n = 3).
Figure 5
Figure 5
Flow cytometry analysis of northeast red bean hemagglutinin-treated MCF7 cells upon (A) Annexin V-PI staining and (B) JC-1 staining.
Figure 6
Figure 6
Cell cycle analysis of MCF7 cells treated with different concentrations of northeast red bean hemagglutinin.
Figure 7
Figure 7
Hoechst 33258 staining of MCF7 cells treated with northeast red bean hemagglutinin.

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