The Mechanisms Of A Novel Agonistic Anti-human Dr5 Monoclonal Antibody Inducing Apoptosis On Jurkat And U937 Cells

BackgroundsTumor necrosis factor( TNF)-related apoptosis-inducing ligand( TRAIL) is a member of the TNF superfamily with the ability to induce apoptosis in a wide variety of transformed cell lines of diverse origin. At least five receptors for TRAIL have been identified so far .Two of them, DR4 (TRAIL-R1)and DR5(TRAIL-R2),are capable of transducing an apoptosis signal, whereas the other three, DcRl(TRAIL-R3), DcR2(TRAIL-R4) and osteoprotegerin(OPG),serve as decoy receptors to block TRAIL - mediated apoptosis. DR4 and DR5 share a common intracellular domain, called the death domain(DD), which is indispensable for initiation of the intracellular signaling leading to cell death. TRAIL triggers multiple cell signals,including the activation of apoptotic caspase cascade,c-JunN -terminal kinase(JNK),p38 mitogen-activated protein kinase(MAPK) and NF-κB.In contrast to TNFs and Fas ligand( FasL),TRAIL has been known to induce apoptosis in a variety of tumor cells and some viral infected cells but not in most normal cells. The potential and safety of soluble TRAIL (sTRAIL) as an anticancer therapeutic agent has been demonstrated in mice and non-human primates. In addition to sTRAIL,monoclonal antibodies(mAbs) against TRAIL receptors with tumoricidal activity are also potential candidates for cancer therapy. There are a number of agonistic mAbs against human DR4 or DR5 reported in previous studies, most of which need crosslinkers to ensure efective killing of tumor cell. However, a recent study showed that at least some anti-DR5 and anti-DR4 mAbs did induce human hepatocytes apoptosis. Therefore, we can not draw a definite conclusion on the hepatocyte toxicity of soluble TRAIL or mAbs against TRAIL receptors by now. Studies on the mechanism of anti-DR5 mAbs will help us to understand the complicated signal pathways mediated by DR5.In the present study,we report studies on mDRA-6,a novel monoclonal antibody against human DR5. mDRA-6 induces apoptosis of various tumor cell lines in the absence of cross-linking in vitro and exhibits a strong tumoricidal activity in vivo. mDRA-6 does not induce cell death of human primary peripheral-blood lymphocytes and normal hepatocytes and does not compete with TRAIL for binding to DR5. Downstream cell signals induced by mDRA-6 were also studied. mDRA-6 activates Caspase cascade and induce a classical apoptosis on Jurkat and U937 cells, and mDRA-6 is capable of activating of JNK,NF-κB and P38. Illustration of the mechanisms may lead to the development of news trategies for cancer immunotherapy. The humanized version of mDRA-6 may be a potential therapeutic agent in expectation in the near future.ObjectivesTo observe the mechanisms of a novel agonistic anti-human DR5 monoclonal antibody inducing apoptosis on Jurkat and U937 cells.MethodsThe DNA fragmentation on Jurkat and U937 cells was analysed by agrose gel electrophoresis. Cell growth rate was detected by MTT assay. The apoptosis of mt)RA-6 on Jurkat and U937 cells and the effects of inhibitors of Caspase-8,-10,-3,-9 and JNK,NF-κB on apoptosis of cells induced with mDRA-6 were detected by flow cytometry with AnnexinV-FITC/PI staining. The expression of Caspase-8,-10,-3,-9,PARP,p-JNK,P-38 and Bax,Bcl-2,Bcl-xL were detected by Western blot.The immunoprecipitation method was used to detect the formation of DISC. The RT-PCR(semi-quantitative reverse Transcription Polymerase chain reaction)method was used to detect the mRNA expression of Caspase-8,Caspase -10 and DR5. Results1.Agrose gel electrophoresis indicated that DNA fragmentation occurred on Jurkat and U937 cells treated with mDRA-6 .while there were no DNA fragmentation on Jurkat and U937 cells which pretreated with Caspase inhibitor.2.MTT analysis found that the rate of cells death induced by mDRA-6 was 59.97% and 42.38% on Jurkat and U937 cells respectively.while the rate of cells death reduced 45.32% (P<0.05) and 4.33%(P>0.05) on Jurkat and U937 cells respectively which pretreated with Caspase-8 inhibitor . the rate of cells death induced by mDRA-6 reduced 4.52%(P>0.05) and 30.36%(P<0.05) on Jurkat and U937 cells respectively which pretreated with Caspase-10 inhibitor.3.The apoptosis rate was 62.62% and 35.65%for Jurkat and U937 cells respectively with mDRA-6 treatment for 2h. The apoptosis rate of Jurkat cells was reduced 45.87%,4.33%,46.44% and 42.75% which pretreated with Caspase-8 inhibitor,Caspase-10 inhibitor,Caspase-9 inhibitor and Caspase inhibitor Z-VAD-FMK; The apoptosis rate of U937 cells was reduced 0.89%,11.67%,2.33% and 1.57% which pretreated with Caspase-8 inhibitor, Caspase-10 inhibitor, Caspase-9 inhibitor and Caspase inhibitor Z-VAD-FMK. The apoptosis rate was increased after the activation of JNK and NF-κB was blocked by SP600125, a JNK inhibitor and BAYll-7082,a NF-κB inhibitor. The apoptosis rate of Jurkat cells was increased 10.63% and 5.14% which pretreated with JNK inhibitor and NF-κB inhibitor; The apoptosis rate of U937 cells was increased to26.67% and 21.1% which pretreated with JNK inhibitor and NF-κB inhibitor.4.Western blotting revealed that Caspase -3,-9,PARP were actived on Jurkat and U937 cells treated with mDRA-6. On U937 cells, Caspase-10 were activated, while on Jurkat cells caspase8 were activated. The activation of Caspase-3 and -9 were all blocked by Caspase inhibitor Z-VAD-FMK. Western blotting exhibited that mDRA-6 induced the activation of p-JNK and P38. The expression of p-JNK was detected 5 minutes after mDRA-6 treatment, while the activation of p-JNK was gradually suppressed with the treatment of Caspase inhibitor Z-VAD-FMK.5.Immunoprecipitation of DISC showed that using mDRA-6, Caspase-10 was detected on U937 cells,while on Jurkat cells Caspase -10 was not detected.6.The mRNA expression of Caspase -8,Caspase -10 and DR5 on Jurkat and U937 cells were analyzed by RT-PCR. The mRNA expression of DR5 and Caspase-8 on Jurkat and U937 cells were no obvious difference(P>0.05).while the mRNA expression of Caspase-10 on U937 cells was higher than Jurkat cells (P<0.05).Conclusions1.mDRA-6 is an agonistic antibody, which induces Jurkat and U937 cells apoptosis through binding to DR5 followed by apoptotic Caspase cascade. In different cells, the apoptosis signal induced by mDRA-6/DR5 were transducted by different initiator Caspases, On Jurkat cells initiator Caspase is Caspase-8, on U937 cells initiator Caspase is Caspase-10. The difference of the mRNA expression of Caspase-10 (P<0.05) between Jurkat and U937 cells may relate to the result that different initiator Caspases were activated on Jurkat and U937 cells.2.mDRA-6-induced apoptosis activated p-JNK and P38,SP600125 inhibits the activation of JNK, NF-κB inhibitor inhibits the activation of NF-κB,and therefore increases the apoptosis rate of Jurkat and U937 cells induced by mDRA-6.