| Galectins are a family of animal lectins that bind to ?-D-galactosides.Galectin-3contains the chimeric structure which is unique in the galectin family,and it has one carbohydrate-recognition domain(CRD)connected to the collagen-repeating N terminal domain.Galectin-3 is a tumor-associated protein,which can promote tumor cell growth,proliferation,adhesion,metastasis and angiogenesis.What's more,galectin-3 induces T cell apoptosis and activation.Thus,it has become the key molecule in the process of tumor immune escape.However,the mechanism of galectin-3 inducing T cell apoptosis remains unclear.Pectin is rich in galacturonic acid,it has anti-tumor activity and immunomodulating activity.Current research indicates that pectin has inhibitory effect on galectin-3.But few work reported pectin exerts the anti-tumor activity through interacting with galectin-3.In this study,the mechanism of galectin-3 inducing T cell apoptosis and activation was investigated.What's more,the active pectins that could affect galectin-3's function were selected,and their anti-tumor activity was studied by using the mice model,to make clear the relationship between the in vitro activity and the in vivo activity of pectins.The results were shown as follows:Firstly,the mechanism of galectin-3 inducing T cell apoptosis was investigated.The apoptotic effect of galectin-3 was confirmed by FITC-Annexin V/PI double staining and by western blotting in both Jurkat and Jurkat E6-1 cells.Moreover,caspase-9 and caspase-3 were activated by galectin-3.What's more,galectin-3 could induce ERK and PKC phosphorylation as well as ROS production.By using the related inhibitors and activators,ROS--MEK-ERK and PKC--MEK-ERK pathways were found.Secondly,the functional domain of galectin-3's apoptotic effect was elucidated by galectin-3 truncated proteins.The first 12 animo acids were disposable,and residues 13-68 and 69-110 were related to ROS--MEK-ERK and PKC--MEK-ERK pathways,respectively.What's more,CRD was essential for the apoptotic activity.Thirdly,the mechanism of galectin-3 inducing T cell activation was explored,by using flow cytometry and ELISA.The results showed that galectin-3 could induce highly expression of CD69 and IL-2 secretion,in time-and dose-dependent manner.Three activation signaling pathways were found: ROS--MEK-ERK,PKC--MEK-ERKand Ras-PI3K-Akt.Fourthly,the relationship between galectin-3 inducing T cell apoptosis and activation was investigated.By treating with different concentrations of galectin-3 on T cells for the indicated time,we found activation occurred prior to apoptosis.As Ras-PI3K-Akt pathway was only found in activation process,activation and apoptosis could occur separately.What's more,the functional domains for galectin-3 triggering apoptosis and activation were same.Finally,the relationship between pectin's inhibitory effect on galectin-3 and their anti-tumor activity was discussed.Functional pectins were selected from 26 types of pectins,and found that RG-I type ginseng pectin WGPA-E-1 and RG-I-4 could inhibit galectin-3 inducing T cell apoptosis,whereas they had little effect on T cell activation.MCP could inhibit both activities.Potato Galactan could not inhibit apoptosis,but it significantly promoted T cell activation.Moreover,WGPA-E-1 and RG-I-4 could inhibit sacaroma S-180 growth,which might because they inhibited T cell apoptosis induced by galectin-3,without affecting T cell activation,prompting more T cells survival and exerting immunofunctions to kill tumor cells.In summary,the signaling pathways for galectin-3 inducing T cell apoptosis and activation were elucidated,and the relationship between apoptosis and activation was clarified.What's more,pectin's selective inhibitory effect on galectin-3 was found corresponding to their anti-tumor effect.This study is important for exploring the mechanism of tumor immune escape and developing pectins as anti-tumor drugs. |
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