Alocasia macrorrhiza rhizome lectin inhibits growth of pathogenic bacteria and human lung cancer cell in vitro and Ehrlich ascites carcinoma cell in vivo in mice.

Cancer and antibiotic resistance represent significant global challenges, affecting public health and healthcare systems worldwide. Lectin, a carbohydrate-binding protein, displays various biological properties, including antimicrobial and anticancer activities. This study focused on anticancer and antibacterial properties of Alocasia macrorrhiza lectin (AML). AML, with a molecular weight of 11.0 ± 1.0 kDa was purified using Ion-exchange chromatography, and the homotetrameric form was detected by gel-filtration chromatography. It agglutinates mouse erythrocytes, that was inhibited by 4-Nitrophenyl-α-d-mannopyranoside. Maximum hemagglutination activity was observed below 60 °C and within a pH range from 8 to 11. Additionally, it exhibited moderate toxicity against brine shrimp nauplii with LD50 values of 321 µg/ml and showed antibacterial activity against Escherichia coli and Shigella dysenteriae. In vitro experiments demonstrated that AML suppressed the proliferation of mice Ehrlich ascites carcinoma (EAC) cells by 35 % and human lung cancer (A549) cells by 40 % at 512 µg/ml concentration. In vivo experiments involved intraperitoneal injection of AML in EAC-bearing mice for five consecutive days at doses of 2.5 and 5.0 mg/kg/day, and the results indicated that AML inhibited EAC cell growth by 37 % and 54 %, respectively. Finally, it can be concluded that AML can be used for further anticancer and antibacterial studies.

Asparagus racemosus and Geodorum densiflorum lectins induce apoptosis in cancer cells by altering proteins and genes expression.

A lectin (designated as ARL) was purified first time from the Asparagus racemosus root with the molecular weight of 14.0 kDa containing about 4.8% carbohydrate. ARL showed hemagglutination activity in both mice and human erythrocytes that were inhibited by three complex sugars among the 26 sugars tested. ARL was thermostable that mostly preserved activity at its optimum pH 8.0. Around 48% and 52.5% human colorectal cancer (HCT-116) cells growth was inhibited by 160 µg/ml of ARL and 256 µg/ml of previously purified Geodorum densiflorum rhizome lectin (GDL). Induction of apoptosis in HCT-116 cells was confirmed by Hoechst 33342 staining, caspase inhibitors, but ROS generation was only observed for ARL. The expression level of BAX and p53 genes increased with a decrease of PARP gene expression for both lectins. The expression of FAS and FADD were increased with the decrease of WNT after treatment with GDL. ARL inhibited 68% and 26% of Ehrlich ascites carcinoma cell growth in vivo in mice after treating with 3.0 and 1.5 mg/kg/day doses for five consecutive days. ARL increased the expression level of NFκB and arrested S cell cycle phase in EAC cells, in contrast, G2/M phase was arrested by ARL and GDL in HCT-116.

Zizyphus mauritiana Fruit Extract-Mediated Synthesized Silver/Silver Chloride Nanoparticles Retain Antimicrobial Activity and Induce Apoptosis in MCF-7 Cells through the Fas Pathway.

Recently, green synthesis of silver/silver chloride nanoparticles (Ag/AgCl-NPs) has gained a lot of interest because of the usage of natural resources, rapidness, eco-friendliness, and benignancy. Several researchers reported that silver-based biogenic NPs have both antimicrobial and anticancer properties. In the present study, Ag/AgCl-NPs were synthesized from Zizyphus mauritiana fruit extract, and their antibacterial, antifungal, and antiproliferative mechanisms against human MCF-7 cell lines were evaluated. Synthesis of Ag/AgCl-NPs from the Z. mauritiana fruit extract was confirmed by the changes of color and a peak of the UV-visible spectrum at 428 nm. The nanoparticles were characterized by transmission electron microscopy, energy dispersive X-ray, X-ray powder diffraction, thermal gravimetric analysis, atomic force microscope, and Fourier transform infrared. Antibacterial activity was checked against four pathogenic bacteria and two fungi. Cytotoxicity was checked against human breast cancer cell line (MCF-7) and mice Ehrlich ascites carcinoma (EAC) cells by MTS assay and clonogenicity assay. Cell morphology of the control and nanoparticle-treated MCF-7 cells were checked by Hoechst 33342, YF488-Annexin V, and caspase-3 substrates. The level of reactive oxygen species (ROS) was studied by using 2',7'-dichlorofluorescein-diacetate staining. Real-time polymerase chain reaction was used for gene expression. Synthesized nanoparticles were heat stable cubic crystals with an average size of 16 nm that contain silver and chlorine with various functional groups. The synthesized Ag/AgCl-NPs inhibited the growth of three pathogenic bacteria (Bacillus subtilis, Shigella boydii, and Escherichia coli) and two fungi (Aspergillus niger and Trichoderma spp.). Ag/AgCl-NPs inhibited the growth of MCF-7 and EAC cells with the IC50 values of 28 and 84 µg/mL, respectively. No colony was formed in MCF-7 cells in the presence of these nanoparticles as compared with control. Ag/AgCl-NPs induced apoptosis and generated ROS in MCF-7 cells. The expression level of FAS, FADD, and caspase-8 genes increased several folds with the decrease of PARP gene expression. These results demonstrated that the anti-proliferation activity of Ag/AgCl-NPs against MCF-7 cells resulted through ROS generation and induction of apoptosis through the Fas-mediated pathway.

Moringa oleifera seed lectin inhibits Ehrlich ascites carcinoma cell growth by inducing apoptosis through the regulation of Bak and NF-κB gene expression.

A Moringa oleifera seed lectin (MOSL) was purified by using chitin column with the molecular mass of 17±1kDa. The lectin agglutinated mouse, cow and human erythrocytes and the hemagglutination activity was inhibited by methyl-α-d-mannopyranoside, methyl-ß-d-galactopyranoside, lactose and glucose. The lectin exhibited 100% hemagglutination activity at the pH range from 8.0 to 9.0 and temperature range from 30 to 60°C. Additionally, the lectin gradually lost its activity in the presence of urea but the activity abolish completely when treated with EDTA. MOSL showed mild toxicity against brine shrimp nauplii with a LC50 value of 131.0µg/ml. Antiproliferative activity was studied against Ehrlich ascites carcinoma (EAC) cells and 71.08% cell growth inhibition was observed in vitro at 200µg/ml. The lectin was injected (i.p.) into EAC mice at the doses of 2.0 and 4.0mg/kg/day for five consecutive days and 25.38% and 55% of cell growth inhibition was observed, respectively. MOSL caused the cell cycle arrest at G2/M phase as determined by FACS flow cytometry. The cell growth inhibition was due to the induction of apoptosis in the EAC cells which was confirmed by cell morphological study, caspase-3 inhibitor and activation of Bak and suppression of Bcl-2 and NF-κB genes expression.

Purification and characterization of a Ca(2+)-dependent novel lectin from Nymphaea nouchali tuber with antiproliferative activities.

A lectin (termed NNTL) was purified from the extracts of Nymphaea nouchali tuber followed by anion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on HiTrap Phenyl HP and by repeated anion-exchange chromatography on HiTrap Q FF column. The molecular mass of the purified lectin was 27.0 ± 1.0 kDa, as estimated by SDS/PAGE both in the presence and in the absence of 2-mercaptoethanol. NNTL was an o-nitrophenyl ß-D-galactopyranoside sugar-specific lectin that agglutinated rat, chicken and different groups of human blood cells and exhibited high agglutination activity over the pH range 5-9 and temperatures of 30-60 °C. The N-terminal sequence of NNTL did not show sequence similarity with any other lectin and the amino acid analysis revealed that NNTL was rich in leucine, methionine and glycine residues. NNTL was a glycoprotein containing 8% neutral sugar and showed toxicity against brine shrimp nauplii with an LC(50) value of 120 ± 29 µg/ml and exerted strong agglutination activity against four pathogenic bacteria (Bacillus subtilis, Sarcina lutea, Shigella shiga and Shigella sonnei). In addition, antiproliferative activity of this lectin against EAC (Ehrlich ascites carcinoma) cells showed 56% and 76% inhibition in vivo in mice at 1.5 and 3 mg·kg(-1)·day(-1) respectively. NNTL was a divalent ion-dependent glycoprotein, which lost its activity markedly in the presence of denaturants. Furthermore, measurement of fluorescence spectra in the presence and absence of urea and CaCl(2) indicated the requirement of Ca(2+) for the stability of NNTL.

Origin of mechanical strength of bovine carbonic anhydrase studied by molecular dynamics simulation.

The forced unfolding process of bovine carbonic anhydrase II (BCA II) was examined at the atomic level by the molecular dynamics (MD) simulation. By force spectroscopy, experimentally obtained force-extension curves (F-E curves) showed a prominent force peak after 50 nm extension. F-E curves obtained from our simulation had three force peaks appearing after extensions of 10-17 nm, 40 nm, and 53 nm, each signifying a brittle fracture of a specific local structure. Upon undergoing the final fracture at 53 nm of extension, the entire molecule became a single flexible chain and was further extended to its full theoretical length, almost as a random coil. This feature of the 53-nm peak strongly suggested its close correspondence to the experimentally observed force peak at approximately 60-nm extension. The 53-nm peak in the molecular dynamics simulation corresponded to the unfolding process of the beta-sheeted core that includes zinc-coordinating histidine residues. These results suggest that the structural change occurring at 50-60 nm in atomic force microscopy experiments corresponded to the destruction of the zinc coordination site.

Dynamics of a partially stretched protein molecule studied using an atomic force microscope.

The dynamics of a single protein molecule subjected to forced mechanical unfolding was investigated in a millisecond time domain using a custom-made atomic force microscope (AFM) apparatus, which allows simultaneous measurements of an average tensile force applied to a single molecule and its mechanical response with respect to an external oscillation. Our target protein was genetically engineered bovine carbonic anhydrase II (BCA) which is a monomeric globular protein, and it has been shown that the as-expressed BCA from Escherichia coli contains two conformational isomers, one with enzymatic activity (type I) and the other without (type II). An interesting feature observed from the dynamic measurements was that when the type I BCA conformer was extended, it often exhibited a clear out-of-phase response against an external oscillation. The type II BCA conformer, however, always exhibited an in-phase response to the external oscillation. This relationship between different types of BCA and their dynamical behaviors was evidently observed around the discontinuous transition point from type I to II.

The importance of being knotted: effects of the C-terminal knot structure on enzymatic and mechanical properties of bovine carbonic anhydrase II.

In order to better understand the contribution of the knotted folding pattern to the enzymatic and mechanical properties of carbonic anhydrases, we replaced Gln-253 of bovine carbonic anhydrase II with Cys, which allowed us to measure the mechanical strength of the protein against tensile deformation by avoiding knot tightening. The expressed protein, to our surprise, turned out to contain two conformational isomers, one capable of binding an enzymatic inhibitor and the other not, which led to their separation through affinity chromatography. In near- and far-UV circular dichroism and fluorescence spectra, the separated conformers were very similar to each other and to the wild-type enzyme, indicating that they both had native-like conformations. We describe new evidence which supports the notion that the difference between the two conformers is likely to be related to the completeness of the C-terminal knot formation.