Purification and cloning of lectin that induce cell apoptosis from Allium chinense.

ABSTRACT

A 8.7 kDa lectin with high agglutin activity was isolated by affinity chromatography and cloned from Allium chinense in this study. For the MTT assay, approximately 60 µg/ml A. chinense lectin (ACL) inhibited 50% of the human hepatoma Hep-3B cells grown after 48 h. In addition, no antiproliferative effect was observed on normal human umbilical vein endothelial cells (HUVEC) even at 100 µg/ml concentration. After treatments with ACL on Hep-3B cells, morphologic changes in the nucleus and cytoskeleton were observed under laser scanning confocal microscopy with 4',6-diamidino-2-phenylindole and tubulin Alexa Fluor 488 staining; whereas, the mitochondrial membrane potential was observed through Mito Tracker Red CMXRos staining. The results showed that ACL led to cell morphology and structure change (e.g., round cell shrinkage). Moreover, ACL resulted in significant change in the shape of the nucleus, damaged the cytoskeleton when tubulin was degraded, and reduced the mitochondrial transmembrane potential. By contrast, no changes were observed on HUVEC cells under the same treatment conditions. DNA fragmentation analysis was used to detect DNA damage. Western blot showed that ACL upregulated caspase-3 and Bax expression during apoptosis and cloned the structural gene of ACL with an open reading frame of 456 bp encoding 151 amino acid residues. The results showed that ACL is a potential anticancer drug.