Lectin from green speckled lentil seeds (Lens culinaris) triggered apoptosis in nasopharyngeal carcinoma cell lines

Abstract

Background: The green speckled lentil seed (Lens culinaris) lectin (GSLL) exhibits hemagglutinating activity, and possesses some properties distinct from those of other lentil lectins (e.g., molecular size, biological activities) that deserve further investigation. This study aims to investigate the basic properties (e.g., molecular size, amino acid sequence, sugar specificity) and biological activities (e.g., antiproliferative activity) of GSLL.

Methods: GSLL was purified by successive fractionation on SP-Sepharose, Affi-gel blue gel, Mono Q, and Superdex 75. The biochemical properties of GSLL were investigated by SDS-PAGE, mass spectrometry, N-terminal amino acid sequencing, and sugar inhibition tests. For the biological activities, purified lyophilized GSLL was sterilized, adjusted to concentrations from 1 to 0 mg/mL (by twofold serial dilution) in Dulbecco's modified Eagle's medium with fetal bovine serum, and examined by using the MTT assay, flow cytometry, and western blotting after treatment of nasopharyngeal carcinoma CNE1 and CNE2 cell lines with the lectin.

Results: GSLL appeared as a 21-kDa band in non-reducing SDS-PAGE. It was composed of two subunits with molecular sizes of 17 and ~4 kDa. It exhibited specificity in binding to glucose and mannose, as well as glucosides and mannosides. Mass spectrometry and N-terminal amino acid sequencing revealed similarity of GSLL to L. culinaris lectin (LcL), especially higher coverage of the β-chain of LcL. A 48-h treatment with GSLL exerted antiproliferative effects on nasopharyngeal carcinoma CNE1 and CNE2 cell lines with significant inhibition at 0.125 mg/mL (P < 0.001) and 1 mg/mL (P = 0.004), respectively, and these effects were attenuated in the presence of glucose and mannose. GSLL induced apoptosis in nasopharyngeal carcinoma CNE1 cells, with detectable phosphatidylserine externalization, mitochondrial depolarization, and cell cycle arrest. Western blot analysis suggested that GSLL triggered the extrinsic apoptotic pathway involving caspase 3, 8, and 9 in CNE1 cells.

Conclusion: GSLL possessed some different properties from LcL (e.g., lower pI), and increased caspase 3, 8, and 9 activity in CNE1 cells.

Fig. 1

Elution profile for purification of GSLL through a Mono Q and b Superdex 75

Fig. 2

a Results of reducing SDS-PAGE of fractions from various steps of purification using a 15 % polyacrylamide gel. Lane 1 crude seed extract; lane 2 Affi-gel blue gel, bound fraction; lane 3 Mono Q, first bound peak, purified lectin from Superdex 75, last peak; lane M protein ladder. b Results of non-reducing SDS-PAGE of GSLL using a 15 % polyacrylamide tricine gel. Lane 5 GSLL; lane M protein ladder

Fig. 3

Effects of glucose, mannose, and glucosamine on the hemagglutinating activity of GSLL. Increases in the concentrations of the specific sugars of GSLL strengthened the effects of competitive inhibition on the hemagglutinating activity. The tests were performed three times

Fig. 4

Results of the MTT assay for GSLL-treated a CNE1 and b CNE2 cells for 24, 48, or 72 h, and c GSLL-treated CNE1 cells under co-treatment with 62.5 mM glucose or mannose for 48 h. Data represent mean ± SD (n = 3). The marked data points represent those with significant differences of P < 0.05: a *P < 0.001, **P = 0.004, ***P = 0.0082, ****P = 0.0095, *****P = 0.0113; b *P < 0.001, **P = 0.004, ***P = 0.044; c *P < 0.001, **P = 0.036, ***P = 0.043

Fig. 5

Results of flow cytometry for 48-h GSLL-treated CNE1 cells examined for a phosphatidylserine externalization (with annexin V-FITC and PI staining), b mitochondrial depolarization (with JC-1 staining), and c cell cycle analysis (with PI staining)

Fig. 6

Results of western blotting analysis for 48-h GSLL-treated CNE1 cells