Antitumor Activity of a Lectin Purified from Punica granatum Pulps against Ehrlich Ascites Carcinoma (EAC) Cells.

BACKGROUND: Lectins are carbohydrate-binding proteins with various pharmacological activities, such as antimicrobial, antidiabetic, antioxidant, and anticancer. Punica granatum fruit extract has traditional uses, however, the anti-cancer activity of purified lectin isolated from P. granatum pulp is yet to be reported. OBJECTIVE: The goals of this study are purification, characterization of the lectin from P. granatum, and examination of the purified lectin's anticancer potential. METHODS: Diethylaminoethyl (DEAE) ion-exchange chromatography was used to purify the lectin, and SDSPAGE was used to check the purity and homogeneity of the lectin. Spectrometric and chemical analysis were used to characterize the lectin. The anticancer activity of the lectin was examined using in vivo and in vitro functional assays. RESULTS: A lectin, designated as PgL of 28.0 ± 1.0 kDa molecular mass, was isolated and purified from the pulps of P. granatum and the lectin contains 40% sugar. Also, it is a bivalent ion-dependent lectin and lost its 75% activity in the presence of urea (8M). The lectin agglutinated blood cells of humans and rats, and sugar molecules such as 4-nitrophenyl-α-D-manopyranoside and 2- nitrophenyl -ß- D-glucopyranoside inhibited PgL's hemagglutination activity. At pH ranges of 6.0-8.0 and temperature ranges of 30°C -80°C, PgL exhibited the highest agglutination activity. In vitro MTT assay showed that PgL inhibited Ehrlich ascites carcinoma (EAC) cell growth in a dose-dependent manner. PgL exhibited 39 % and 58.52 % growth inhibition of EAC cells in the mice model at 1.5 and 3.0 mg/kg/day (i.p.), respectively. In addition, PgL significantly increased the survival time (32.0 % and 49.3 %) of EAC-bearing mice at 1.5 and 3.0 mg/kg/day doses (i.p.), respectively, in comparison to untreated EAC-bearing animals (p < 0.01). Also, PgL reduced the tumor weight of EAC-bearing mice (66.6 versus 39.13%; p < 0.01) at the dose of 3.0 mg/kg/day treatment. Furthermore, supplementation of PgL restored the haematological parameters toward normal levels deteriorated in EAC-bearing animals by the toxicity of EAC cells. CONCLUSION: The results indicated that the purified lectin has anticancer activity and has the potential to be developed as an effective chemotherapy agent.

Trichosanthes dioica seed lectin inhibits Ehrlich ascites carcinoma cells growth in vivo in mice by inducing G0 /G1 cell cycle arrest.

Trichosanthes dioica seed lectin (TDSL), having a molecular mass of 57 ± 2 kDa was purified in an alternative way. For the purification process, the galactose-sepharose-4B affinity column was used. The purified TDSL agglutinated human and mouse erythrocytes at the minimum concentration of 8  µg/ml. d-lactose and d-galactose were the most potent inhibitory sugars as observed. The purified lectin was a glycoprotein having 3.0% of a neutral sugar. The lectin exhibited maximum activity up to 60°C and pH range from 7.0 to 10.0 and stable up to 4.0 M urea as tested. The lectin demonstrated mild toxicity when administered against brine shrimp nauplii, and the LC50 value was calculated to be 84.0 µg/ml. Minimum agglutination of Ehrlich ascites carcinoma (EAC) cells caused by the lectin was found at the protein concentration of 1.56 µg/ml. TDSL inhibited 7, 50.2%, and 60.3% of the EAC cells growth in vivo in mice when administered with 0.75, 1.5, and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. After lectin treatment, red blood cell (RBC) and hemoglobin levels were increased significantly toward the normal compared with EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 of the lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase, which was observed using flow cytometry. In conclusion, TDSL can be a candidate for the potent anticancer agents that exerts low toxicity toward brine shrimp nauplii. PRACTICAL APPLICATIONS: A 57 ± 2 kDa lectin (designated TDSL) was purified from Trichosanthes dioica seeds using a galactose-sepharose-4B affinity column. The lectin demonstrated mild toxicity and agglutinated Ehrlich ascites carcinoma (EAC) cells. The lectin inhibited 50.2% and 60.3% of the EAC cell growth in vivo in mice when administered with 1.5 and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. The lectin increased RBC and hemoglobin level toward the normal compared with lectin-treated EAC cells-bearing, EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase. The lectin can be a candidate for potent anticancer agents.

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 novel chitinase from Trichosanthes dioica seed with antifungal activity.

Chitinases are a group of enzymes that show differences in their molecular structure, substrate specificity, and catalytic mechanism and widely found in organisms like bacteria, yeasts, fungi, arthropods actinomycetes, plants and humans. A novel chitinase enzyme (designated as TDSC) was purified from Trichosanthes dioica seed with a molecular mass of 39±1 kDa in the presence and absence of ß-mercaptoethanol. The enzyme was a glycoprotein in nature containing 8% neutral sugar. The N-terminal sequence was determined to be EINGGGA which did not match with other proteins. Amino acid analysis performed by LC-MS revealed that the protein was rich in leucine. The enzyme was stable at a wide range of pH (5.0-11.0) and temperature (30-90 °C). Chitinase activity was little bit inhibited in the presence of chelating agent EDTA (ethylenediaminetetraaceticacid), urea and Ca(2+). A strong fluorescence quenching effect was found when dithiothreitol and sodium dodecyl sulfate were added to the enzyme. TDSC showed antifungal activity against Aspergillus niger and Trichoderma sp. as tested by MTT assay and disc diffusion method.