Anti-Neuroblastoma Properties of a Recombinant Sunflower Lectin

Abstract

According to their sugar recognition specificity, plant lectins are proposed as bioactive proteins with potential in cancer treatment and diagnosis. Helja is a mannose-specific jacalin-like lectin from sunflower which was shown to inhibit the growth of certain fungi. Here, we report its recombinant expression in a prokaryotic system and its activity in neurobalstoma cells. Helja coding sequence was fused to the pET-32 EK/LIC, the enterokinase/Ligation-independent cloning vector and a 35 kDa protein was obtained in Escherichia coli representing Helja coupled to thioredoxin (Trx). The identity of this protein was verified using anti-Helja antibodies. This chimera, named Trx-rHelja, was enriched in the soluble bacterial extracts and was purified using Ni⁺²-Sepharose and d-mannose-agarose chromatography. Trx-rHelja and the enterokinase-released recombinant Helja (rHelja) both displayed toxicity on human SH-SY5Y neuroblastomas. rHelja decreased the viability of these tumor cells by 75% according to the tetrazolium reduction assay, and microscopic analyses revealed that the cell morphology was disturbed. Thus, the stellate cells of the monolayer became spheroids and were isolated. Our results indicate that rHelja is a promising tool for the development of diagnostic or therapeutic methods for neuroblastoma cells, the most common solid tumors in childhood.

Keywords: Helianthus annuus; Helja; agglutinin; jacalin; neuroblastoma; recombinant lectin; tumor toxicity.

Figure 1

Induction of recombinant Trx-rHelja expression. The protein profile of extracts was obtained after lysis of cells from un-induced (−) or isopropyl β-d-1-thiogalactopyranoside (IPTG) induced (+) E. coli cultures. The extracts obtained from 0.1 mL of cell cultures with an OD at 600 nm of 1.5 were loaded on a 12% denaturing polyacrylamide gel electrophoresis, SDS-PAGE, and subsequently stained with Coomassie Brillant Blue (A). Immunodetection of Trx-rHelja in extracts of 0.01 mL of un-induced (−) or IPTG induced (+) cell cultures. Gel performed as described above was transferred to nitrocellulose, blocked and incubated sequentially with 1:3000 anti-Helja polyclonal antibodies and alkaline phosphatase-conjugated anti-rabbit IgG 1:10,000 (B). Figures are representative of three (n = 3) independent experiments. Molecular mass markers are indicated on the left.

Figure 2

Recombinant Trx-rHelja purification by affinity chromatography. The protein profile of samples collected during Trx-rHelja purification scheme was analyzed using sequentially affinity for Ni²⁺-Sepharose and mannose-agarose matrixes. The soluble fraction (SN) of extracts from 100 mL IPTG induced E. coli cultures were loaded on the metal affinity matrix and the non-retained (NR) or the retained (R) fractions were recovered. Then, R was loaded on a mannose-agarose column and the proteins, the non-retained (NRm), and retained peaks (Rm) were recovered. Aliquots of R, NR, Rm and NRm fractions were analyzed in 12% SDS-PAGE and subsequently stained with Coomassie Brillant Blue. Figure is representative of two (n = 2) independent experiments. Molecular mass markers are indicated on the left.

Figure 3

Trx-rHelja digestion with enterokinase and rHelja purification. Aliquots of the mix containing enterokinase after (+) and before (−) of the incubation were loaded on 12% SDS-PAGE and subsequently transferred to nitrocellulose, blocked and incubated sequentially with 1:3000 anti Helja polyclonal antibodies and 1:10,000 alkaline phosphatase-conjugated anti-rabbit IgG (A). Aliquots of the proteins from the non-retained (NR) or the retained (R) peaks obtained after loading the products of the enterokinase digestion on the Ni²⁺-Sepharose matrix were analysed in a 12% SDS-PAGE and stained with Coomassie Brillant Blue (B) or transferred to nitrocellulose, blocked and incubated sequentially with 1:3000 anti Helja polyclonal antibodies and 1:10,000 alkaline phosphatase-conjugated anti-rabbit IgG (C). Figures are representative of three (n = 3) independent experiments. Molecular mass markers are indicated on the left.

Figure 4

Cell viability of human SH-SY5Y neuroblastoma cells treated with Trx-rHelja or rHelja. The cells at density of 5 × 10⁴ cell/well were grown for 24 h before the incubation with 6 and 12 µM Trx-rHelja or rHelja. After an additional 24 h, MTT reagent was added to the wells. Dimethyl sulfoxide was used to dissolve the formazan complex and the absorbance was measured at 570 nm. The cellular viability was expressed as a percentage of the value obtained in the absence of treatment. Each experiment was tested in triplicates and they were repeated at least three times. Standard deviation, one-way ANOVA and Tukey test analysis (p ≤ 0.05) were performed using InfoStat software (2014, Agricultural College of the National University of Córdoba, Córdoba, Argentine). Different capital letters indicate significant differences.

Figure 5

Cell morphology changes in human neuroblastoma SH-SY5Y cells treated with Trx-rHelja or rHelja. The cells at density of 5 × 10⁴ cell/well were grown for 24 h before the incubation with 6 and 12 µM Trx-rHelja or rHelja for additional 24 h. Cells were examined using a phase contrast microscopy (10× lens). Scale bar = 10 μm.