Tepary Bean Lectin Research Articles

EGFR and p38MAPK Contribute to the Apoptotic Effect of the Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) in Colon Cancer Cells.

Previous works showed that a Tepary bean lectin fraction (TBLF) induced apoptosis on colon cancer cells and inhibited early colonic tumorigenesis. One Tepary bean (TB) lectin was expressed in Pichia pastoris (rTBL-1), exhibiting similarities to one native lectin, where its molecular structure and in silico recognition of cancer-type N-glycoconjugates were confirmed. This work aimed to determine whether rTBL-1 retained its bioactive properties and if its apoptotic effect was related to EGFR pathways by studying its cytotoxic effect on colon cancer cells. Similar apoptotic effects of rTBL-1 with respect to TBLF were observed for cleaved PARP-1 and caspase 3, and cell cycle G0/G1 arrest and decreased S phase were observed for both treatments. Apoptosis induction on SW-480 cells was confirmed by testing HA2X, p53 phosphorylation, nuclear fragmentation, and apoptotic bodies. rTBL-1 increased EGFR phosphorylation but also its degradation by the lysosomal route. Phospho-p38 increased in a concentration- and time-dependent manner, matching apoptotic markers, and STAT1 showed activation after rTBL-1 treatment. The results show that part of the rTBL-1 mechanism of action is related to p38 MAPK signaling. Future work will focus further on the target molecules of this recombinant lectin against colon cancer.

Comparative study, homology modelling and molecular docking with cancer associated glycans of two non-fetuin-binding Tepary bean lectins.

We present the purification and characterization of the two most abundant isoforms of lectins isolated from Tepary bean (Phaseolus acutifolius) seeds, which have been shown to differentially affect the survival of different cancer cells. They were separated by concanavalin A-affinity chromatography. After purification, to release the N-glycans, they were digested with the endoglycosidases PNGase and Glycanase A. Fractions resulted from the hydrolysis products were analyzed to determine their carbohydrate composition. Mass spectrometry data indicated that both isoforms contained high mannose glycans being mannose 6 the most abundant form. Furthermore, based on sequence Ans-X-Ser/Thr, where X is any amino acid except proline, a glycosylation site was determined on asparagine 36. When their metal requirement to preserve their biological activity was determined, the lectins showed differences. While lectin A (LA) agglutination activity was best in the presence of magnesium, lectin B (LB) was best with calcium. Additionally, only LA exhibited affinity to human type-A erythrocytes. Although both lectins showed small differences in their properties, an identical structure-model for both lectins was generated by the homology modelling process. Also, the analysis of ligand binding sites and in silico glycosylation were achieved. Molecular docking with colon adenocarcinoma associated-N-glycans revealed some highly possible interactions and, on the other hand, that N-glycan interaction zones of Tepary bean lectins is not restricted to the carbohydrate binding domain but to an extended part of their surface, which could lead new strategies to explain their biological activity.

Protein Concentrates on Tepary Bean (Phaseolus acutifolius Gray) as a Functional Ingredient: In silico Docking of Tepary Bean Lectin to Peroxisome Proliferator-Activated Receptor Gamma.

The tepary bean (Phaseolus acutifolius Gray) is a US–Mexico frontier native crop, produces high yields in agriculture, and needs to be reconsidered because of its nutritional and functional properties. This study aimed to determine the technological and nutritional properties of flours and protein concentrates of tepary bean, besides determining an in silico agonist effect of tepary bean lectin to peroxisome proliferator-activated receptor gamma (PPAR-γ). We evaluated the technological properties of raw samples (tepary flour and tepary protein concentrate) and cooked samples (tepary flour and tepary protein concentrate). The flours present a significant difference (p < 0.05) concerning protein concentrates in water absorption and oil absorption capacity. The raw samples' emulsifying capacity was higher than that reported in the literature for other legumes, but not the cooked samples. The samples' foaming capacity had no significant difference in treatments (p > 0.05), and cooked tepary bean protein concentrate presented complete gelation at a lower concentration (2%). Nutritionally, raw samples present a protein percentage of 23.46 ± 0.06 and 71.38 ± 0.44 and cooked samples present a protein percentage of 25.27 ± 0.04 and 62.69 ± 0.14; a chemical score of 72, 86, 82, and 72; in vitro protein digestibility (%) = 48.20 ± 0.31, 49.80 ± 0.80, 61.77 ± 1.70, and 63.61 ± 4.19; and C-PER = 0.86, 1.34, 1.93, and 1.81, respectively. All the samples showed methionine + cysteine as the limiting amino acid. All these nutritional data are very similar to the common bean (Phaseolus vulgaris). SDS-PAGE preserves the lectin fraction in both protein concentrates. The in silico study of tepary lectin (PDB: 6tt9) shows that there were seven peptides that presented values below −120 kcal/mol: PEW, VSVGF, PSQK, TTPW, ATSF, ITY, and TSF, with VSVGF, PSQK, and PEW having the highest affinity for active sites of the PAPRγ receptor (binding energies from −5.32 to −7.04 kcal/mol). These peptides could show antiadipogenic or antidiabetic activity based on the intermolecular bond energies and open an interesting research item.

Bioaccessibility and In Vitro Intestinal Permeability of a Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) Using the Everted Intestine Assay.

Tepary bean (Phaseolus acutifolius) lectins exhibit differential in vitro and in vivo biological effects, but their gastrointestinal interactions and digestion have not yet been assessed. This work aimed to evaluate the changes of a recombinant Tepary bean lectin (rTBL-1) through an in vitro and ex vivo gastrointestinal process. A polyclonal antibody was developed to selectively detect rTBL-1 by Western blot (WB) and immunohistochemical analysis. Everted gut sac viability was confirmed until 60 min, where protein bioaccessibility, apparent permeability coefficient, and efflux ratio showed rTBL-1 partial digestion and absorption. Immunoblot assays suggested rTBL-1 internalization, since the lectin was detected in the digestible fraction. The immunohistochemical assay detected rTBL-1 presence at the apical side of the small intestine, potentially due to the interaction with the intestinal cell membrane. The in silico interactions between rTBL-1 and some saccharides or derivatives showed high binding affinity to sialic acid (−6.70 kcal/mol) and N-acetylglucosamine (−6.10 kcal/mol). The ultra-high-performance liquid chromatography–electron spray ionization–quantitative time-of-flight coupled to mass spectrometry (UHPLC–ESI–QTOF/MS) analysis showed rTBL-1 presence in the gastric content and the non-digestible fraction after intestinal simulation conditions. The results indicated that rTBL-1 partially resisted the digestive conditions and interacted with the intestinal membrane, whereas its digestion allowed the absorption or internalization of the protein or the derivative peptides. Further purification of digestion samples should be conducted to identify intact rTBL-1 protein and digested peptides to assess their physiological effects.

Recombinant Lectin from Tepary Bean (Phaseolus acutifolius) with Specific Recognition for Cancer-Associated Glycans: Production, Structural Characterization, and Target Identification.

Herein, we report the production of a recombinant Tepary bean lectin (rTBL-1), its three-dimensional (3D) structure, and its differential recognition for cancer-type glycoconjugates. rTBL-1 was expressed in Pichia pastoris, yielding 316 mg per liter of culture, and was purified by nickel affinity chromatography. Characterization of the protein showed that rTBL-1 is a stable 120 kDa homo-tetramer folded as a canonical leguminous lectin with two divalent cations (Ca2+ and Mn2+) attached to each subunit, confirmed in its 3D structure solved by X-ray diffraction at 1.9 Å resolution. Monomers also presented a ~2.5 kDa N-linked glycan located on the opposite face of the binding pocket. It does not participate in carbohydrate recognition but contributes to the stabilization of the interfaces between protomers. Screening for potential rTBL-1 targets by glycan array identified 14 positive binders, all of which correspond to β1-6 branched N-glycans’ characteristics of cancer cells. The presence of α1-6 core fucose, also tumor-associated, improved carbohydrate recognition. rTBL-1 affinity for a broad spectrum of mono- and disaccharides was evaluated by isothermal titration calorimetry (ITC); however, no interaction was detected, corroborating that carbohydrate recognition is highly specific and requires larger ligands for binding. This would explain the differential recognition between healthy and cancer cells by Tepary bean lectins.

Tepary Bean (Phaseolus acutifolius) Lectins Induce Apoptosis and Cell Arrest in G0/G1 by P53(Ser46) Phosphorylation in Colon Cancer Cells.

A Tepary bean lectin fraction (TBLF) has been studied because it exhibits differential cytotoxic and anticancer effects on colon cancer. The present work focuses on the evaluation of the apoptotic mechanism of action on colon cancer cells. Initially, lethal concentrations (LC50) were obtained for the three studied cell lines (HT-29, RKO and SW-480). HT-29 showed the highest LC50, 10 and 100 times higher than that of RKO and SW-480 cells, respectively. Apoptosis was evaluated by flow cytometry, where HT-29 cells showed the highest levels of early and total apoptosis, caspases activity was confirmed and necrosis was discarded. The effect on cell cycle arrest was shown in the G0/G1 phase. Specific apoptosis-related gene expression was determined, where an increase in p53 and a decrease in Bcl-2 were observed. Expression of p53 gene showed the maximum level at 8 h with an important decrease at 12 and 24 h, also the phosphorylated p53(ser46) increased at 8 h. Our results show that TBLF induces apoptosis in colon cancer cells by p-p53(ser46) involvement. Further studies will focus on studying the specific signal transduction pathway.

Effects of intragastrically-administered Tepary bean lectins on digestive and immune organs: Preclinical evaluation.

Previous work showed that Tepary bean (Phaseolus acutifolius) lectins exhibit differential cytotoxic effects on cancer cell lines by apoptosis induction. In vivo studies using a Tepary bean lectin fraction (TBLF, 50 mg/kg of body weight) after colon cancer induction in rats showed that TBLF inhibited early precancerous lesions without systemic toxicity however, loss of body weight gain and activation of immune cells were observed. In order to know more about the possible adverse effects, we evaluated the administration of TBLF on digestive and immune organs. Sprague Dawley rats were administered TBLF for six weeks and allowed to recover for two weeks. Immune activation was observed through an increased lymphocyte-granulocyte ratio, an increased number of lymphoid follicles in intestinal Peyer's patches and a slight expansion of the splenic white pulp. Atrophy was observed in small intestine villi and crypt foci of the colon without normalization after the recovery period. Pancreas histopathology showed hypertrophy after the six-week administration period, particularly vacuolation and trabecular widening; but after the two-week recovery period atrophy was observed, suggesting a partial compensatory type process. Our results show that TBLF activates the immune system and affects digestive organs through direct interaction with intestinal epithelium, and indirectly by producing pancreatic hyperfunction. Further work will focus in longer recuperation periods after TBLF treatment.

Phaseolus acutifolius Lectin Fractions Exhibit Apoptotic Effects on Colon Cancer: Preclinical Studies Using Dimethilhydrazine or Azoxi-Methane as Cancer Induction Agents.

Phaseolus acutifolius (Tepary bean) lectins have been studied as cytotoxic molecules on colon cancer cells. The toxicological profile of a Tepary bean lectin fraction (TBLF) has shown low toxicity in experimental animals; exhibiting anti-nutritional effects such as a reduction in body weight gain and a decrease in food intake when using a dose of 50 mg/kg on alternate days for six weeks. Taking this information into account, the focus of this work was to evaluate the effect of the TBLF on colon cancer using 1,2-dimethylhydrazine (DMH) or azoxy-methane/dextran sodium sulfate (AOM/DSS) as colon cancer inductors. Rats were treated with DMH or AOM/DSS and then administered with TBFL (50 mg/kg) for six weeks. TBLF significantly decreased early tumorigenesis triggered by DMH by 70%, but without any evidence of an apoptotic effect. In an independent experiment, AOM/DSS was used to generate aberrant cryptic foci, which decreased by 50% after TBLF treatment. TBLF exhibited antiproliferative and proapoptotic effects related to a decrease of the signal transduction pathway protein Akt in its activated form and an increase of caspase 3 activity, but not to p53 activation. Further studies will deepen our knowledge of specific apoptosis pathways and cellular stress processes such as oxidative damage.

Cytotoxic and antiproliferative effect of tepary bean lectins on C33-A, MCF-7, SKNSH, and SW480 cell lines.

For many years, several studies have been employing lectin from vegetables in order to prove its toxic effect on various cell lines. In this work, we analyzed the cytotoxic, antiproliferative, and post-incubatory effect of pure tepary bean lectins on four lines of malignant cells: C33-A; MCF-7; SKNSH, and SW480. The tests were carried out employing MTT and 3[H]-thymidine assays. The results showed that after 24 h of lectin exposure, the cells lines showed a dose-dependent cytotoxic effect, the effect being higher on MCF-7, while C33-A showed the highest resistance. Cell proliferation studies showed that the toxic effect induced by lectins is higher even when lectins are removed, and in fact, the inhibition of proliferation continues after 48 h. Due to the use of two techniques to analyze the cytotoxic and antiproliferative effect, differences were observed in the results, which can be explained by the fact that one technique is based on metabolic reactions, while the other is based on the 3[H]-thymidine incorporated in DNA by cells under division. These results allow concluding that lectins exert a cytotoxic effect after 24 h of exposure, exhibiting a dose-dependent effect. In some cases, the cytotoxic effect is higher even when the lectins are eliminated, however, in other cases, the cells showed a proliferative effect.

Tepary bean lectins induces apoptosis on human colon cancer cells and inhibits early tumorogenesis in rats

The aim of this study was to determine whether a lectin fraction (LF) from Tepary bean seeds (Phaseolus acutifolius) exhibits apoptotic induction on colon cancer cells and its in vivo effect on tumorogenesis in rats. Median lethal concentration (0.402 mg protein/mL) on HT‐29 cells caused a significant increase in caspase 3 activity, an overexpression of caspase 3 (35%) and caspase 9 (78%) and a 36.6% apoptosis increase determined by flow cytometry. Colon cancer was induced on male Sprague Dawley rats treated for 8 weeks with 1,2‐dimethylhydrazine (DMH) (40 mg/body weight kg) and a subsequent administration of 50 mg of LF/body weight kg for 6 weeks. The LF provoked a decrease in food consumption and body weight of rats with a partial recovery at the end of the experiment. Histopathological analysis showed that LF decreased the incidence of early stages tumors in a 53% but no effect was observed on high grade carcinogenesis. The results show that LF induces apoptosis in colon cancer and it has inhibitory effect on early tumorogenesis.Founding: CONACYT Ciencia Básica (82349)

Purification, Biochemical Characterization, and Bioactive Properties of a Lectin Purified from the Seeds of White Tepary Bean (Phaseolus Acutifolius Variety Latifolius)

The present work shows the characterization of Phaseolus acutifolius variety latifolius, on which little research has been published, and provides detailed information on the corresponding lectin. This protein was purified from a semi-domesticated line of white tepary beans from Sonora, Mexico, by precipitation of the aqueous extract with ammonium sulfate, followed by affinity chromatography on an immobilized fetuin matrix. MALDI TOF analysis of Phaseolus acutifolius agglutinin (PAA) showed that this lectin is composed of monomers with molecular weights ranging between 28 and 31 kDa. At high salt concentrations, PAA forms a dimer of 63 kDa, but at low salt concentrations, the subunits form a tetramer. Analysis of PAA on 2D-PAGE showed that there are mainly three types of subunits with isoelectric points of 4.2, 4.4, and 4.5. The partial sequence obtained by LC/MS/MS of tryptic fragments from the PAA subunits showed 90–100% identity with subunits from genus Phaseolus lectins in previous reports. The tepary bean lectin showed lower hemagglutination activity than Phaseolus vulgaris hemagglutinin (PHA-E) toward trypsinized human A and O type erythrocytes. The hemagglutination activity was inhibited by N-glycans from glycoproteins. Affinity chromatography with the immobilized PAA showed a high affinity to glycopeptides from thyroglobulin, which also has N-glycans with a high content of N-acetylglucosamine. PAA showed less mitogenic activity toward human lymphocytes than PHA-L and Con A. The cytotoxicity of PAA was determined by employing three clones of the 3T3 cell line, demonstrating variability among the clones as follows: T4 (DI50 51.5 µg/mL); J20 (DI50 275 µg/mL), and N5 (DI50 72.5 µg/mL).

Detection of Cytotoxic Activity of Lectin on Human Colon Adenocarcinoma (Sw480) and Epithelial Cervical Carcinoma (C33-A)

Lectins comprise a heterogeneous class of proteins that recognize the carbohydrate moieties of glycoconjugates with high specificity. Numerous studies have shown that lectins are capable of recognizing specific carbohydrate moieties displayed by malignant cells or tissues. The present work was performed to investigate the effects of tepary bean (Phaseolus acutifolius) lectins on proliferation, colony formation, and alteration of DNA synthesis of human malignant cells. Tepary bean lectin showed dose dependent effects on the inhibition of viability as well as on colony formation in two human malignant cells lines (C33-A, Sw480); By contrast, tepary bean lectin only showed significant effects on DNA synthesis on Sw480 cells. Our results provide evidence of the anti- proliferative and cytotoxic effects of the tepary bean lectins on C33-A and Sw480 cells lines.

Cytotoxic effect of a tepary bean (Phaseolus acutifolius) lectin on human cancer cell lines

Lectins can specifically recognize carbohydrates of cell membranes and some of them exhibit anticancer properties. In a previous work, we identified a cytotoxic lectin from tepary bean with differential antiproliferative effect on normal and transformed fibroblasts. The goal of the present work was to evaluate the effect of a concentrated tepary bean lectin fraction (TBLF) on human cancer cell lines growth. TBLF was obtained by molecular weight exclusion and ion exchange chromatography and it was tested on breast cancer cells (MCF‐7 and ZR‐75), cervix cancer cells (HeLa, SiHa and C33A) and colon cancer cells (CaCo2). All cell lines showed a cytotoxic effect, after 72 h of treatment. Inhibitory (IC50) and lethal (LC50) concentrations were calculated for each cell line. The most sensible cells were colon cancer cells with IC50 of 0.119 and LC50 of 3.0 μg protein/mL. CaCo2 and normal intestinal cells from rat illeon (IEC‐18), included as normal counterpart, were also tested at 24 h, LC50 did not change for CaCo2 but IC50 was of 0.563 μg protein/mL; for IEC‐18, IC50 and LC50 were of 0.993 and 7.95 μg protein/mL, respectively. Our results show that TBLF is able to recognize selectively different types of cancer cells, more over between normal and cancer cells. Current studies are focusing on TBLF mechanism action and on in vivo studies against colon cancer.

Acute toxicity evaluation of a tepary bean (Phaseolus acutifolius) lectin

As plant lectins have shown anticancer potential, we had previously tested the cytotoxic effect of a lectin from tepary bean on human cancer cells proliferation and the most sensible were colon cancer cells. Before testing the lectin against colon cancer in vivo, it is necessary to determine its toxicity, therefore the goal of the present work was to evaluate the acute toxicity of a concentrated tepary bean lectin fraction (TBLF). Female Sprague Dawley rats of 7 weeks old were administrated with the TBLF at doses of 5, 50, 300 and 2000 mg/kg body weight (423 OECD) by intragastric canula. Food intake was determined daily and body weight each 5 days, until 15 days. Highest doses caused diarrhea, lethargy and piloerection in the first 24 h, but a full recovery was observed after 48 h. After necropsy, no alterations were observed in liver, stomach, gut, colon, lungs, heart, thymus and spleen; no alteration was observed in the colon histology. Lectin adminstration increased leucocytes in 54% with 50 mg/kg (Tukey, p&lt;0.05) and 2000 mg/kg decreased the number in 25%. TBLF by oral administration showed typical signs of lectins toxicity but it was not lethal and animals had a full recovery. Current studies are focusing on the evaluation of chronic toxicity.

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety "Red Kidney."