Antiproliferative Activity of Sword Bean Lectin and Carbonate Apatite Against Ehrlich Ascites Carcinoma Cell

Abstract

A glucose specific lectin has been purified from the seeds of Canavalia gladiata (sword bean) in a single step by affinity chromatographic technique. For protein extraction, delipidated sword bean meal was homogenized with 100 mM sodium acetate-acetic acid buffer pH 5.5 for 5 hours at 4-6oC. The clear supernatant was separated by centrifugation at 11,000 rpm for 10 minutes at 4-6oC and designated as crude extract. The crude extract was subjected to 90% ammonium sulfate precipitation for overnight at 4-6oC. After centrifuge at 11,000 rpm for 10 minutes 4-6oC, the precipitate was dissolved in 100mM sodium acetate-acetic acid buffer pH 5.5 containing 1mM MnCl2, 1mM CaCl2 and 100mM NaCl. The protein solution was dialyzed against the same buffer to remove remaining ammonium sulfate. After dialysis, the sample was centrifuged and the clear supernatant was injected to the affinity column packed with Sephadex G-200 gel and equilibrated with binding buffer (100mM sodium acetate-acetic acid buffer pH 5.5 containing 1mM MnCl2, 1mM CaCl2 and 100mM NaCl). The column was washed to remove unbound materials. Gel-bound protein was eluted with 100mM sodium acetate-acetic acid buffer pH 5.5 containing 0.4M glucose. Finally, in order to remove salts and glucose from the eluted protein solution, it was dialyzed against deionized distilled water. The purity of the protein was checked by 12.5% SDS-PAGE method. The SDS-PAGE gel shows five bands in the lane of crude extract and single band in the lane of purified protein, which indicates the affinity matrix system was able to purify only one kind of protein in a single step. The molecular weight of the affinity purified sword bean lectin (SBL) was determined and it was found to be 28 KDa. The purified sword bean lectin powerfully agglutinate human blood types A, B, O erythrocytes almost equally. The order of hemagglutination activity with chicken and mice erythrocytes was mice>> chicken> human blood types A, B and O groups but not with the bovine and goat erythrocytes. This SBL was stable upto 60°C and showed maximum hemagglutinating activity at pH 7.5. The sugar inhibition assay exhibited its sugar specificity highly against D(+) glucose at Minimal Inhibition Concentration (MIC) of 25 mM and maltose, D(-) mannose, fructose at MIC of 3.125 mM. But lectins were not inhibited by the presence of D(+) galactose, L(+) arabinose. Antiproliferation activity of purified SBL was tested against Ehrlich Ascites Carcinoma (EAC) cell in the swiss albino mice at a dose of 4 mg/kg/day and it worked as an active inhibitor. The result was compared with a known effective anticancer drug, bleomycin at the dose of 0.3 mg/kg/day (i.p.). The cell growth inhibition was found to be 77% and 90% for SBL and bleomycin respectively. RBC level of EAC cell bearing mice increased to 8.6 which is very close to normal value of 9.4. The hemoglobin level also increased to 10.0 which is near to normal value of 11.4 gm/dl. Carrier of various drugs and bio-active molecules such as peptides, proteins and DNAs etc. through the cell membrane into cells has attracted increasing attention because of its importance in medicine and drug delivery. Therefore, SBL combining with inorganic carbonate apatite as drug carrier in different concentration was also used to treat EAC cell bearing mice to get a better result, but the result was not satisfactory. The combined effect of SBL and carbonate apatite decreased EAC cell growth inhibition from 77% to 47% and 45% to 9% when 5mM carbonate apatite (4 ml/kg/day) was used with SBL at the dose of 4mg/kg/day and 2.5 mg/kg/day respectively whereas 5mM carbonate apatite at the same dose itself gave a very good inhibition of 70%. The RBC and hemoglobin level increased after treating with carbonate apatite to 8.0 and 10.9 which is nearer to normal level of 9.4 and 11.4 respectively. Fluorescence study of SBL and SBL combined with carbonate apatite showed that surface induced denaturation of SBL was occurred when it was administered in combination with carbonate apatite. As a result it prohibit the individual antiproliferation effect of sword bean lectin and carbonate apatite.