Study On The Mechanism Of RIP3 Induced Apoptosis In Tumor Cells

RIP3,a member of receptor interaction protein family,is low/or no expression in tumor cells.Our previous study found that recombinant mung bean trypsin inhibitor LBBI can activate the expression of RIP3 in tumor cells,and induce the apoptosis of tumor cells.Therefore,we speculated that the expression of RIP3 in tumor cell can directly induce tumor cells apoptosis,but the mechanism is still unclear.There are several domains in RIP3 kinase,which contains protein kinase domain,RHIM,NES,NLS and other domains.In our study,we would explore the domain which plays role in cell apoptosis and how apoptosis are activated by RIP3,moreover we also explored how RIP3 were induced to express in tumor cells by recombinant LBBI.Finally,we would understand the mechanism of tumor cells apoptosis induced by recombinant LBBI.The analysis of RIP3' structure was carried on bioinformatics software.There are three NES between the site of 100 and 200 at RIP3' N terminal.According to the results,there might be a NES domain amino acid residues in the 300,while the typical NLS was failed to be find in RIP3.Besides those domain,there is also a RHIM in RIP3 C terminal from 417 aa to the end.Three NES domains in N terminal was deleted by PCR method,and RHIM was deleted by the same method.Those mutations confirmed that the deletion of NES in N terminal didn't effect on the apoptosis of MCF-7 cells,and RHIM in RIP3 play role in cell apoptosis.Further studies showed that RIP3 and N terminal deletion mutants can activate Caspase3,Caspase9,Caspase8 and induce cell apoptosis.Apoptosis could be induced and the expression of RIP3 was activated when MCF-7 cells.We also confirmed that LBBI could down-regulate the expression of KLK6 and up-regulate the expression of IGFBP3 in MCF-7 cells.Over expression of RIP3 confirmed that RIP3 did not affect the expression of KLK6 in MCF-7 cells.Furthermore,overexpression of IGFBP3 could up-regulate the expression of RIP3 in MCF-7 cells.Based on the above results,we believe that cell apoptosis would occur when RIP3 was expressed highly in MCF-7 cells,this process of apoptosis requires the RHIM domain and the presence of at least a nuclear export signal.Recombinant mung bean LBBI induce MCF-7 to apoptosis through its inhibition of the expression and secretion of KLK6,and up-regulation of IGFBP3 expression,the activation of RIP3,and this apoptosis happened in MCF-7 cells via Caspase pathway.Therefore,RIP3 may be a therapeutic target in the treatment of malignant tumors,and it provides a new way for tumor therapy.