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. 2020 Jul 4;5(27):16430-16439.
doi: 10.1021/acsomega.0c00577. eCollection 2020 Jul 14.

Biochemical Characterization of Lactose Binding Entadin Lectin from Entada rheedii Seeds with Cytotoxic Activity against Cancer Cell Lines

Sanjay Naik  1 , Sanjit Kumar  1
Affiliations

Biochemical Characterization of Lactose Binding Entadin Lectin from Entada rheedii Seeds with Cytotoxic Activity against Cancer Cell Lines

Sanjay Naik et al. ACS Omega. .

Abstract

A novel Entadin lectin was isolated, purified, and characterized from the seeds of Entada rheedii by ammonium sulfate precipitation, followed by lactose affinity chromatography. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified Entadin lectin appeared as a single band (monomeric in nature) with a molecular mass of approximately 20 kDa in both reducing and nonreducing conditions. Mass spectroscopic analysis confirms the molecular weight of Entadin lectin as 19,333 Da. Entadin lectin showed a highest titer value in agglutination against human blood group B red blood cells, and its hemagglutination activity was inhibited by lactose, cellobiose, and galactose. Periodic acid Schiff staining confirmed the glycoprotein nature of Entadin lectin with an approximately 5% carbohydrate content. This lectin is highly stable even after incubation at a wide range of temperatures (30-60 °C) and pHs (6-10). The antiproliferative effect of Entadin lectin against lung cancer cells A549 and cervical cancer cells HeLa showed IC50 values of 28 and 32 μg/mL, respectively, and no antiproliferative activity against normal cells was observed. Cell morphological studies revealed that Entadin lectin induced apoptosis in both A549 and HeLa cancer cells, which was confirmed by acridine orange/ethidium bromide and Hoechst (33258) nuclear counterstaining.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Analysis of purified Entadin lectin using a lactose affinity column: (LI) molecular weight marker, (LII) crude extract of E. rheedii seeds, (LIII) purified Entadin lectin shows 20 kDa on SDS-PAGE, and (LIV) silver staining of lactose column-purified Entadin lectin.
Figure 2
Figure 2
Mass spectra analysis of intact Entadin lectin through LC–ESI-MS.
Figure 3
Figure 3
(A) Hemagglutination activity of purified Entadin lectin using human O, B, and A blood cells. (B) PAS staining: L1—ovalbumin, L2—Entadin lectin.
Figure 4
Figure 4
Biophysical properties of Entadin lectin: (A) temperature stability at different temperatures and (B) pH stability at different pHs.
Figure 5
Figure 5
Effect of monovalent, divalent, and trivalent metal ions on the hemagglutination activity of Entadin lectin.
Figure 6
Figure 6
Spectroscopic studies of Entadin lectin at various temperatures.
Figure 7
Figure 7
Dose-dependent cytotoxic effect (MTT) of Entadin lectin on HeLa, A549, and Vero cells.
Figure 8
Figure 8
Morphological characterization of cells treated with Entadin lectin further stained with AO/EtBr (A) human lung cancer cell line (A-549) and (B) human cervical cancer cell line (HeLa) with varying concentrations: (a) control, (b) 0.5× IC50 μg/mL, (c) 1× IC50 μg/mL, (d) 2× IC50 μg/mL, and (e) cells treated with doxorubicin considered as a positive control.
Figure 9
Figure 9
(A and B) Morphological observation of treated human lung cancer cell line (A-549) and human cervical cancer cell line (HeLa) with different lectin concentrations: (1) control, (2) 0.5× IC50 μg/mL, (3) 1× IC50 μg/mL, (4) 2× IC50 μg/mL, and (5) positive control treated with doxorubicin further stained with Hoechst stain.
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