Role Of MG132, A Proteasome Inhibitor, In Inducing Expression Of Costimulatory Molecules CD86, Signal Transduced Pathway, Apoptosis Pathway In Leukemic Cells And Its Effect Of Allogeneic Mixed Lymphocyte Reaction

Objective To investigate the role of proteasome inhibitors MG132 in the inducing costimulatory molecules CD80 and CD86 expression, signal transduced pathway, apoptosis and pathway in leukemic cells and its effect of allogeneic mixed lymphocyte reaction .Methods Acute myelocytic leukemia cells of the line HL-60 and chronic myelocytic leukemia cells of the line K562 were cultured. 7-AAD staining and flow cytometry (FC) were used to examined the viability of the cells. MG132, a proteasome inhibitors, of the concentrations of 2 or 3μmol/L was added into the culture fluid of HL-60 cells for 24h and 48h respectively and then annexinⅤ/7-AAD staining and FC were used to detect the apoptosis of the apoptosis of the cells. HL-60 and K562 cells treated with 1μmol/L MG132 for 24h and 48h respectively, anti-CD80 and anti-CD86 antibodies were added, then FC was used to detect the expression of CD80 and CD86. The mRNA expression of CD86 in the HL-60 cells treated with 1μmol/L MG132 was examined by RT-PCR. Protein kinase inhibitor G?6976,JNK inhibitor SP600125, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK,histone deacetylase inhibitor Trichostatin A and p21 and p27 MAPK inhibitor Apigenin blocked protein kinase pathway, JNK pathway caspase-8 pathway, caspase-9 pathway, histone acetylase pathway and p21 and p27 MAPK pathway in MG-132-inducing the expression of CD86 in HL-60 cells by Flow Cytometer measuring. caspase-8 inhibitor Z-IETD-FMK and caspase-9 inhibitor Z-LEHD-FMK blocked caspase-8 pathway and caspase-9 pathway in MG132-inducing apoptosis in HL-60 cells by Flow Cytometer measuring. The expression of CD86 mRNA analyzed by RT-PCR after Protein kinase inhibitor G?6976, JNK inhibitor SP600125, caspase-8 inhibitor Z-IETD-FMK, caspase-9 inhibitor Z-LEHD-FMK,histone deacetylase inhibitor Trichostatin A and p21 and p27 MAPK inhibitor Apigenin blocked protein kinase pathway, JNK pathway caspase-8 pathway, caspase-9 pathway, histone acetylase patway and p21 and p27 MAPK pathway in MG132-induced the expression of CD86 in HL-60 cells. HL-60 and K562 cells were treated by 1μmol/L MG132 for 48h and then underwent irradiation of 75 Gy Co-60 to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNC) of healthy volunteers, as reactive cells, were isolated and inoculated into the Co-60 treated HL-60 and K562 cells of different concentrations, as stimulating cells, for 5d, CCK-8, a new agent to detect the cell viability, was added for 4h, and then the A value of absorbance was measured at the wave length of 450 nm of enzyme labeling instrument. Control groups were setn up for all tests.Results The cell viability rates of the HL-60 cell treated with 1μmol/L Mg132 for 24h and 48h were 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 were increased dose- and time-dependently. Before MG132 treatment K562 cells did not express CD86, and the CD86 expression of the HL-60 cells was up-regulated time-dependently (all P<0.01) treatment with low-dosage MG132. The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P<0.001). The expression of CD80 was unchanged. The expression of CD86 was decreased in HL-60 cells after p21 and p27 MAPK inhibitor Apigenin blocked p21 and p27 MAPK pathway treatment with MG132. It showed that MG132-induced the expression of CD86 may via p21 and p27 MAPK pathway. Low-dose MG132 induced the expression of CD86 in HL-60 cells. No significant changes were observed in MG132-induced the expression of CD86 in HL-60 cells after inhibiting protein kinase pathway, JNK pathway, histone acetylase pathway, caspase-8 pathway and caspase-9 pathway. High-dose MG132 directly killed HL-60 cells and induced apoptosis in HL-60 cells. No significant changes were observed in MG132-induced apoptosis after inhibiting caspase-8 and caspase-9 pathway. The expression of p21 protein and p27 protein increased in MG132-induced apoptosis. The expression of p53 protein was unchanging in MG-132-induced apoptosis in HL-60 cells. CKK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×105(P<0.01).No remarkable proliferation of PBMNC was seen in the K562 groups no matter if the HL-60 cells had been treated with MG132.Conclusion Low-dose protesome inhibitor MG132 induces the expression of costimulatory molecular CD86 expression in HL-60 cells, and associated with p21 and p27 MAPK pathway but not associated with protein kinase pathway, JNK pathway, histone acetylase patway, caspase-8 pathway and caspase-9 pathway, improves the proliferation of PBMNCs. High-dose MG132 induced apoptosis and directly killed HL-60 cells. MG132-induced apoptosis via caspase-8- and caspase-9-independent pathway. P21 protein and p27 protein was involved in MG132-induced apoptosis in HL-60. No significant changes were observed in MG132-induced the expression of p53 protein in HL-60 cells. Low-dose MG132 improved the proliferation of PBMNCs from healthy volunteers and enhanced immunity.