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

Yau Sang Chan, Huimin Yu, Lixin Xia, Tzi Bun Ng

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