A lectin with highly potent inhibitory activity toward breast cancer cells from edible tubers of Dioscorea opposita cv. nagaimo

Abstract

A 70-kDa galactose-specific lectin was purified from the tubers of Dioscorea opposita cv. nagaimo. The purification involved three chromatographic steps: anion exchange chromatography on a Q-Sepharose column, FPLC-anion exchange chromatography on a Mono Q column, and FPLC-gel filtration on a Superdex 75 column. The purified nagaimo lectin presented as a single 35-kDa band in reducing SDS-PAGE while it exhibited a 70-kDa single band in non-reducing SDS-PAGE suggesting its dimeric nature. Nagaimo lectin displayed moderate thermostability, retaining full hemagglutinating activity after heating up to 62°C for 30 minutes. It also manifested stability over a wide pH range from pH 2 to 13. Nagaimo lectin was a galactose-specific lectin, as evidenced by binding with galactose and galactose-containing sugars such as lactose and raffinose. The minimum concentration of galactose, lactose and raffinose required to exert an inhibitory effect on hemagglutinating activity of nagaimo lectin was 20 mM, 5 mM and 40 mM, respectively. Nagaimo lectin inhibited the growth of some cancer cell lines including breast cancer MCF7 cells, hepatoma HepG2 cells and nasopharyngeal carcinoma CNE2 cells, with IC(50) values of 3.71 µM, 7.12 µM and 19.79 µM, respectively, after 24 hour treatment with nagaimo lectin. The induction of phosphatidylserine externalization and mitochondrial depolarization indicated that nagaimo lectin evoked apoptosis in MCF7 cells. However, the anti-proliferative activity of nagaimo lectin was not blocked by application of galactose, signifying that the activity was not related to the carbohydrate binding specificity of the lectin.

Figure 1. Profile of elution in purification of nagaimo lectin.

Elution profile of (A) crude nagaimo tuber extract from Q-Sepharose, (B) Fraction II from Mono Q, and (C) Fraction V from Superdex 75. The shaded regions represent the fractions with hemagglutinating activity that were collected in each step.

Figure 2. Results of SDS-APGE.

SDS-PAGE of (A) crude nagaimo tuber extract, (B) Fraction II from Q-Sepharose, (C) Fraction V from Mono Q, (D) Purified nagaimo lectin from Superdex 75, and (E) Purified nagaimo lectin without addition of the reducing agent, β-mercaptoethanol. Lane 1 of each panel: Molecular weight markers (GE Healthcare) including phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.4 kDa). Lane 2 of each panel: nagaimo protein samples.

Figure 3. Effect of (A) different temperatures, (B) different pH, and (C) specific carbohydrates on hemagglutinating activity of Nagaimo lectin.

Figure 4. Results of MTT assay on different cell lines.

Results of MTT assay of MCF7, HepG2 and CNE2 cells treated with different concentrations of nagaimo lectin. Results represent mean±SD (n = 3).

Figure 5. Results of MTT assay of (A) MCF7 cells and (B) HepG2 cells treated with nagaimo lectin, with or without concurrent administration of 100 mM galactose.

The presence of 100 mM galactose could not curtail the retarding effect of nagaimo lectin on the growth of MCF7 and HepG2 cells. Results represent mean±SD (n = 3).

Figure 6. Results of MTT assay of MCF7 cells treated with nagaimo lectin that were pre-treated at different (A) temperatures and (B) pH values for 30 minutes.

The presence of 100 mM galactose could not curtail the retarding effect of Nagaimo lectin on the growth of MCF7 and HepG2 cells. Results represent mean±SD (n = 3).

Figure 7. Flow cytometry analysis on Annexin V-FITC and PI staining.

Flow cytometry analysis of MCF7 cells after treatment with nagaimo lectin (A) with or (B) without the presence of 100 mM nagaimo lectin for 24 hours, followed by staining with Annexin V-FITC.

Figure 8. Flow cytometry analysis on JC-1 staining.

Flow cytometry analysis of MCF7 cells after treatment with nagaimo lectin (A) with or (B) without the presence of 100 mM nagaimo lectin for 24 hours, followed by staining with JC-1.