Leukemia Cell Apoptosis Induced By Anti-hdr5 Monoclonal Antibody And DR5 Expression On Leukaemia Cell

BackgroundTumor 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, TRAIL-R1 (DR4) and TRAIL-R2 (DR5), sharing a commom intracellular domain, called the death domain (DD), which is indispensable for initiation of the intracellular signaling cascade leading to cell death, are capable of transducing an apoptosis signal, whereas the other three, TRAIL-R3 (DcR1), TRAIL-R4 (DcR2) and osteoprotegerin (OPG), serve as decoy receptors to block TRAIL-mediated apoptosis. TRAIL has been known to induce apoptosis in a variety of tumor cells and some virally infected cells but not in most normal cells. However, increasing experimental evidences on hrTRAIL-inducing apoptosis of normal cells(especially hepatocytes) were reported in recent studies, arguing against the potential usefulness and safety of soluble TRAIL in cancer therapy. In addition to sTRAIL, monoclonal antibodies (mAbs) against TRAIL-R1 (DR4) or TRAIL-R2 (DR5) 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 effective killing of tumor cells.We have cloned the human DR5 gene and prepared the soluble recombinant human DR5 protein (rhDR5), established hybridomas and produced an anti-hDR5 monoclonal antibody, mDRA-6. mDRA-6 showed strong tumoricidal activity in the absence of cross-linking, and had different sensitivity for tumor cells.ObjectiveThis study is to detect DR5 expression in leukaemia cells, and evaluate the correlation of DR5 expression with apoptosis induced by mDRA-6 in human leukemia cell. The synergistic effect of mDRA-6 and Vitamin E Succinate (VES) on Raji and K562 cells and its possible mechanism were studied.MethodsCytotoxic and apoptotic effects of mDRA-6 on Jurkat, U937, HL-60, Raji and K562 cells and the synergistic effect of mDRA-6 and vitamin E succinate (VES) on Raji and K562 cells were detected by MTT and flow cytometry with AnnexinV-FITC/PI staining. DR5 expression on leukaemic cell surface was determined by flow cytometric analysis of the binding of anti-DR5 mAb; DR5 total protein expression in leukaemic cells were detected by western blot. The RT-PCR(semi-quantitative reverse transcription polymerase chain reaction)method was used to detect the DR5 mRNA expression in leukaemic cells.Results①The specific cytotoxicity of the mDRA-6 against Jurkat cells was 88.25%, U937 cells was 61.43%,HL-60 cells was 59.76%, Raji cells was 21.98%, and K562 cells was 5.23% respectively at the concentration of 10mg/L for 12 hours by MTT analysis. The combination of mDRA-6 and VES exhibited synergistic effect on Raji cells and K562 cells. 10μg/mL of mDRA-6 and 20μmol/L of VES killed 40.22% Raji cells and 8.67% K562 cells.②The apoptotic rate of the Jurkat, U937, HL-60, Raji and K562 cells was 50.22%, 48.11%, 23.40% and 7.20%, respectively ,in the presence 5mg/L mDRA-6 for 12 hours determined by flow cytometry with Annexin V-FITC/PI staining. The apoptotic rate of Raji and K562 celsl was increased to 43.18% and 16.99% respectively, after treated with 2μg/mL of mDRA-6 and 10μmol/L of VES for 16 hours.③The DR5 expression level on leukaemia cell surface were 94.8% for Jurkat cells, 74.71% for U937 cells, 58.66% for HL-60 cells ,42.35%for Raji cells and 14.92% for K562 cells respectively. The DR5 expression level of Raji cells and K562 cells treated with 10μmol/L of VES for 12 hours increased to 70.08% and 16.38% respectively.④Western blotting revealed that expression levels of the DR5 protein in Jurkat, U937, HL-60, Raji and K562 cells were unequal.The DR5 protein expression levels in Raji cells and K562 cells were lower. However, the DR5 protein levels of Raji cells and K562 cells treated with VES increased .⑤The mRNA expression of DR5 in Jurkat,U937, HL-60, Raji and K562 cells were analyzed by RT-PCR. The mRNA expression of DR5 in Raji cells treated with 20μmol/L of VES for 12 hours increased (P<0.05) obviously.Conclusion①The expression level of DR5 in Jurkat, U937, HL-60, Raji and K562 cells were different. There is the correlation between DR5 expression and human leukemia apoptotic rate induced by mDRA-6 in human leukaemia cell.②Combination of mDRA-6 and vitamin E succinate has synergistic effect of apoptosis on Raji and K562 cells by increasing DR5 expression on Raji, K562 cells treated with vitamin E succinate.