ABSTRACT
Objective To partially purify the toxic factor secreted by A. corymbifera and to analyze the mechanism of A. corymbifera-induced human umbilical vein endothelial cell (HUVEC) apoptosis. Methods Glycoprotein secreted by A. corymbifera was purified by Con A Lectin chromatography. The influence of different protein fractions on HUVEC apoptosis was determined by flow eytometer. Both denaturing and nondenaturing deglycosylation of purified glycoprotein was performed and the ability of the protein moiety and carbohydrate moiety to induce HUVEC apoptosis was evaluated respectively. Activation of related caspases during A. corymbifera-induced apoptosis was analyzed by Western blot. The role of caspase-8 and -9 in HUVEC apoptosis was investigated using caspase inhibitors. Caspase inhibitors were used to stop the suppression of HUVEC viability by XTT assay. Results Flow cytometric analysis shows the total protein as well as the glycoprotein fraction of A. corymbifera may induce HUVEC apoptosis in a dose dependent manner. In contrast, similar activity was not observed in the non-glycoprotein fraction. Neither deglycosylated protein nor carbohydrate moiety is able to induce HUVEC apoptosis alone. In the apoptotic signaling pathway, caspase9, caspase-3 and cytochrome C were activated significantly, except caspase-8. Moreover, caspase-9 inhibitor, instead of caspase-8 inhibitor, completely abrogates A. corymbifera-induced HUVEC apoptosis. Caspase9 and caspase-3 inhibitors completely waived the suppression of HUVEC viability by A. corymbifera. Conclusion Glycoprotein secreted by A. corymbifera is associated with HUVEC apoptosis. Intact glycoprotein is essential for the apoptotic progress. Intrinsic apoptotic signaling pathway mediates A. corymbifera-induced HUVEC apoptosis.
ABSTRACT
Objective To analyze the influence of Absidia corymbifera on cell activity of human umbilical vein endothelial cells (HUVEC) as well as the related mechanism. Methods Time course analy sis of the influence of A. corymbifera on cell viability of HUVEC was determined by cell counting after Trypan blue staining. Apoptosis of HUVEC induced by A. corymbifera was observed under fluorescence microscope after treatment with apoptosis detection kit. Time course analysis of HUVEC apoptosis induced by A. corymbifera was detected by flow cytometry quantitatively. Effect of caspase-3 inhibitor on A. corymbifera associated apoptosis was also evaluated at the same time. Activation of caspase-3 inside HUVEC was detected by Western blot. Results A. corymbifera inhibited cell viability of HUVEC in a time-dependent manner by Trypan blue staining. After 12 hours' co-culture, A. corymbifera began to show suppression on cell viability (P =0. 001 ). Fluorescence microscope observation revealed A. corymbifera induced apoptosis of HUVEC instead of necrosis. Flow cytometry analysis showed A. corymbifera induced apoptosis of HUVEC in a time-dependent manner. A. corymbifera began to show obvious effect on apoptosis after 12 h co-culture (P =0.0036). Moreover, A. corymbifera-associated apoptosis was almost abrogated completely by caspase-3 inhibitor. Western blot analysis demonstrated that A. corymbifera triggered the activation of caspase-3 inside HUVEC in a timedependent fashion. Conclusion A. corymbifera induces apoptosis of HUVEC in vitro. Such apoptotic signal is transmitted through caspase cascade reaction.