ABSTRACT
@#Objective To develop an indirect ELISA-based peptide scanning method combined with nuclear magnetic resonance(NMR)technique for the epitope identification of calcitonin gene-related peptide(CGRP)antibodies.Methods The antigen binding activities of two antibodies(new CGRP antibody and control antibody)were determined by indirect ELISA using each truncated CGRP fragment as coating antigen,and the linear epitope was analyzed according to the EC50value of four-parameter curve. Two-dimensional hydrogen-nitrogen correlation(2D1H-15N HSQC)spectrum of CGRP were acquired by NMR technique,and the binding of antibodies to the arginine of CGRP were analyzed through the disturbance of the antibodies to CGRP signals. Specific arginine modifications were detected by liquid chromatography-mass spectrum(LCMS) and NMR technique,and two arginine resonances were assigned on CGRP by correlating the rank order of the modification rate.ResultsThe antigen binding activities of two antibodies with CGRP(1-37),CGRP(19-37)and CGRP(25-37)showed dose-response relationships,and were fitted with four-parameter equation. However,there were no significant antigen binding with CGRP(1-18),CGRP(19-24)and CGRP(25-37)without C-terminal amide. The linear epitopes of both antibodies were located at the C-terminal of CGRP. The resonances of arginine ε-NH in 2D1H-15N HSQC spectrum disappeared in the presence of the control antibody;and the resonances appeared in the presence of the new antibody. The arginine R11 and R18 of CGRP could bind to the control antibody,but not to the new antibody. The NMR assignment for the arginine resonances were made by correlating the relative ranking of the modification rate where signals A and B arose from R11 ε-NH and R18 ε-NH respectively.ConclusionIn this study,the linear and conformational epitopes of new CGRP antibody and control antibody were identified based on the methods of ELISA and NMR,which may provide a theoretical basis for the design of the candidate therapeutic CGRP antibodies.
ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent.However,emergence of drug resistance limits its potential use.Plumbagin is a natural quinonoid compound isolated from plant.In this study,induced apoptosis effect of the combined treatment with plumbagin and TRAIL on human melanoma A375 cell line was examined and possible mechanism was investigated.The cells were divided into four groups:control group,plumbagin group (plumbagin,5 or 10 μmol/L),TRAIL group (TRAIL,30 ng/mL) and plumbagin+TRAIL group (combined treatment group).The apoptosis,and the expression of DR4 and DR5 were detected by flow cytometry.The activities of caspase-8 and caspase-3 were determined by colorimetric assay.The results showed that the apoptosis rate was 8.3% in TRAIL group,10.35%-16.94% in plumbagin group and 52.39%-55.39% in combined treatment group,respectively,with the difference being significant between combined treatment group and plumbagin or TRAIL group (P＜0.05 for each).Moreover,plumbagin alone could markedly up-regulate DR5 mRNA and protein expression,and slightly increase DR4 mRNA and protein expression.Treatment of human melanoma A375 cells with plumbagin resulted in the activation of Caspase-3,but not Caspase-8.These results suggest that plumbagin might be useful for TRAIL-based treatment for melanoma.
ABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-cancer agent. Epigallocatechin-3-gallate (EGCG) is a polyphenolic constituent of green tea. In this study,inhibitory effect of combined use of EGCG and TRAIL on human melanoma A375 cells was examined and the possible mechanism investigated. The cells were divided into 4 groups:control group,EGCG group (EGCG:10,20 μg/mL),TRAIL group (TRAIL:25 ng/mL) and EGCG+TRAIL group (combined group). The growth inhibition was measured in the A375 cells treated with different concentrations of TRAIL ((25,50,75,100,125,150 ng/mL) by MTT assay. The apoptosis was assessed by flow cytometry. The expressions of DR4 and DR5 were detected by flow cytometry and western blotting. The activities of caspase-8 and caspase-3 were determined by colorimetric assay.The results showed that TRAIL could dose-dependently inhibit the growth of A375 cells and the IC50 of TRAIL was 150 ng/mL. The apoptosis rate was 11.8% in the TRAIL group,5%-7% in the EGCG group and 48.9%-59.1% in the combined group. Significant difference was found in the apoptosis rate between the combined group and the EGCG or TRAIL group (P＜0.05 for each). The expression of DR4 instead of DR5 was significantly increased in the EGCG group. The activity of caspase-3 rather than caspase-8 was substantially enhanced in the EGCG group. These results suggest that EGCG is useful for the TRAIL-based treatment for melanoma.