Caspase-dependent molecular mechanisms of anti-human DR5 monoclonal antibody mDRA-6 inducing apoptosis of human leukemia Jurkat cells.
OBJECTIVE
Both tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and some monoclonal agonistic antibodies against TRAIL receptors have antitumor activity. We have previously prepared a novel monoclonal agonistic antibody against human death receptor 5 (DR5) and designated it as mDRA-6. This study was to explore the Caspase-dependent molecular mechanisms of mDRA-6 inducing apoptosis of human leukemia Jurkat cells.


METHODS
After exposure to different doses of mDRA-6, DNA fragmentation of Jurkat cells was detected by agarose gel electrophoresis, cell proliferation was detected by MTT assay, and cell apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining. Jurkat cells were further treated with the inhibitors for Caspase-10, -9, -8 and -3. The active cleavage products of Caspase-10, -9, -8, -3 and poly ADP-ribose polymerase (PARP), BH3 interacting domain death agonist (Bid), truncated Bid (tBid) and cytochrome c (Cyto c), were analyzed by western blot.


RESULTS
After mDRA-6 treatment, DNA fragmentation was detected in Jurkat cells. mDRA-6 inhibited cell proliferation in a dose-dependent manner. When treated with 2.0 microg/mL mDRA-6, the apoptosis rates of Jurkat cells were 16.2% at 0.25 h, 28.3% at 0.5 h, 69.2% at 1 h and 78.2% at 2 h. Interestingly, the mDRA-6-induced apoptosis was repressed by 77.9% by Caspase-8 inhibitor ZIF, 54.2% by Caspase-3 inhibitor ZDF, and 8.7% by Caspase-9 inhibitor ZLF, but was not repressed by Caspase-10 inhibitor ZAF. After mDRA-6 exposure, the proenzymes of Caspase-8, -9 and -3 were reduced and their active cleavage products were increased along with the increase of exposure time, the cleavage products of PARP were also increased, Bid was degraded to tBid, and an abundance of Cyto c was released from mitochondria, but the proenzyme of Caspase-10 showed no change and no cleavage products of Caspase-10 were detectable.


CONCLUSIONS
mDRA-6 can induce apoptosis of Jurkat cells via the Caspase-dependent and mitochondrial pathways.
Du
Yao-Wu
YW
Institute of Immunology, Medical College, Henan University, Kaifeng, Henan, PR China.
Liu
Guang-Chao
GC
Wang
Jing
J
Zhao
Yue-Ping
YP
Li
Shu-Lian
SL
Chen
Ju-Gao
JG
Jiang
Qi
Q
Cai
Jing
J
Ma
Yuan-Fang
YF
D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D051023:BH3 Interacting Domain Death Agonist Protein / Q000378:metabolism; D015153:Blotting, Western; D053455:Caspase 10 / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D053181:Caspase 8 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects; D045304:Cytochromes c / Q000378:metabolism; D053938:DNA Fragmentation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004587:Electrophoresis, Agar Gel; D005434:Flow Cytometry; D006801:Humans; D019169:Jurkat Cells; D007938:Leukemia / Q000201:enzymology / Q000473:pathology; D009842:Oligopeptides / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000276:immunology*; D013997:Time Factors
D000911:Antibodies, Monoclonal; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D061945:Caspase Inhibitors; D009842:Oligopeptides; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C470795:benzyloxycarbonyl-alanyl-glutamyl-valyl-aspartic acid fluoromethyl ketone; C403753:benzyloxycarbonyl-isoleucyl-glutamyl-threonyl-aspartic acid fluoromethyl ketone; C403754:benzyloxycarbonyl-leucyl-glutamyl-histidyl-aspartic acid fluoromethyl ketone; D045304:Cytochromes c; D011065:Poly(ADP-ribose) Polymerases; D053455:Caspase 10; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases