Characterization of Cytotoxicity against Cancer Cells induced by Human Monoclonal Antibody Specific to TRAIL-R1

PURPOSE: Tumor necrosis facter-related apoptosis-inducing ligand (TRAIL) is a member of the TNF ligand superfamily. Soluble TRAIL(sTRAIL) induces apoptosis in a variety of tumor cells through activation of TRAIL-R1 (DR4) and TRAIL- R2 (DR5) with no effect on most normal cells. Also, Tumor cells that are resistant to TRAIL can be sensitized by combination treatment with chemotherapheutic agent or irradiation. But it has also been reported that sTRAIL is able to induce apoptosis of normal human hepatocyte, and some cancer cells are resistant to sTRAIL treatment. Recently, agonistic antibodies to TRAILR1(DR4) or TRAIL-R2(DR5) have been developed for alternative anti-cancer drug. Although they specifically bound their target receptors and induced apoptosis in a variety of cancer cells, most antibodies developed are mouse antibodies. We generated DR4-4, an agonistic human monoclonal Fab specific to DR4 from human antibody library using phage-display technology. We characterized the Fab and tested possibility of its usage as anti-cancer agents. Also, we tested combined effect of DR4-4 Fab and r-irradiation. MATERIALS AND METHODS: We generated DR4-4 Fab using phage-display technology and the Fab was purified by Ni-NTA affinity chromatography. Purified DR4-4 Fab was confirmed by SDS-PAGE and immunoblotting. Binding specificity of DR4-4 Fab was analyzed by direct ELISA and cytotoxicity of DR4-4 Fab was measured by MTT assay on variety of cancer cells and normal cells. DR4-4 Fab-induced cell death was confirmed by flow cytometry after Annexin-V/PI staining and DNA fragmentation assay. Caspase activation was measured by Apo-ONE caspase3/7 Assay kit, and cleavage of caspase-3 and PARP were detected by immunoblotting. To investigate mechanisms of DR4-4 Fab-induced cytotoxicity, cytotoxicity of DR4-4 Fab was analyzed in various Jurkat mutant cell lines, such as FADD-deficient Jurkat cell lines, JNK-dominant negative (DN), c-JUN-DN, IkB-DN Jurkat cell lines. PI3K inhibitor WM was also used. To investigate the effect of calcium on DR4-4 Fab-induced cell death, Jurkat cells were pretreated with BAPTA/AM (intracellular Ca++ chelator) before treatment of DR4-4 Fab. To determine change of mitochondrial membrane potential, Jurkat cells were treated with DR4-4 Fab and stained with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) and analyzed using flow cytometry. Additionally, To observe morphological changes of the mitochondrial-network in HeLa cells, HeLa cells were treated with DR4-4 and then observed using by confocal microscope(x100) after Hoechst33342 (nucleus) and Mitotracker (mitochondria) staining. Also we tested combination effect, Jurkat cells were treated with DR4-4 Fab and r-irradiation simultaneously and cell death was measured by flow cytometry after Annexin-V/PI staining. RESULTS: The DR4-4 Fab specifically bound to soluble DR4. It induced cell death in various TRAIL-sensitive and TRAIL-resistant cancer cells, but not normal cells. Differently from sTRAIL, DR4-4 Fab showed cytotoxicity against cancer cells in the presence of a pan caspase inhibitor, z-VAD. Activation of caspase-3 and cleavage of PARP were not also observed, although they were shown at a low level at very late stage. Therefore, it is indicated DR4-4 Fab-induced cell death occurs mainly through caspase-independent mechanism. Interestingly, we also found that DR4-4 Fab could induce cell death in c-Jun-DN, JNK-DN, or FADD-deficient jurkat cell lines, but DR4-4 Fab-induced cell death was inhibited by WM(PI3K inhibitor) and BAPTA/AM(intracellular Ca++ chelator). When Jurkat cells were treated with DR4-4 Fab, mitochondrial membrane potential decreased and loss of mitochondria membrane potential was recovered by WM treatment. Additionally, We observed changes in mitochondria network morphology following treatment of HeLa cells with DR4-4 Fab. Also, when Jurkat cells were cotreated with DR4-4 Fab and r-irradiation, cell death was dramatically increased. CONCLUSION: These results suggest that acting mechanisms of the DR4-4 for inducing cytotoxicity on cancer cells are quite different from those of TRAIL and the Fab has a possible potential as a anti-tumor agent even against TRAIL-resistant cancer cells without cytotoxicity to normal cells. Also DR4-4 Fab has potential to be applied in combined therapy with r-irradiation.

Author dc:creator

• 이, 유리

Contributors dc:contributor

• 장, 영주
• 대학원 의학과
• 200624279

• TRAIL 수용체
• agonistic antibody
• 세포독성
• caspase
• 방사선
• TRAIL receptor
• cytotoxicity
• r-irradiaion

Language dc:language

ko

Identifier

http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000005828
000000005828

OAI identifier oai:identifier

oai:repository.ajou.ac.kr:201003/1841