The Effects And The Combined Mechanisms Of Oncolytic Adenovirus ZD55-TRAIL On Multiple Myeloma Cells

Section1:ZD55-TRAIL reduces cell viability and induces apoptosis in multiple myeloma cell lines.Aim:To study cytotoxic effects and apoptosis induced by oncolytic virus ZD55-TRAIL on multiple myeloma cells lines.Methods:Viability of cell growth was measured by MTT assay. The infectivity was measured by flow cytometry. Hoechst staining was used for detecting apoptosis. The expression of Caspase-3, Caspase-8, Caspase-9, PARP and associated proteins in myeloma cell lines were examined by Western blotting.Results:(1) IC50of ZD55-TRAIL on myeloma cell lines RPMI8226、MM1S、MM1R after24h were among3～78MOI, and IC50were among2～6.4MOI after48h. The results showed that ZD55-TRAILwas effective at inducing cytotoxicity in drug-sensitive and -resistant MM cell lines.(2) RPMI8226cells were effectively infected with ZD55-EGFP in a dose-dependent manner under a fluorescent microscopy examination. Flow cytometric analysis showed GFP-positive cells treated with ZD55-EGFP at MOIs of25and50were52%and62%, respectively.(3) The viability of RPMI8226cells for48h was significantly reduced by ZD55-TRAIL in a dose-dependent manner compared with no viral treatment, whereas ZD55had a little cytotoxic effect.(4) IL-6mediates growth and survival of MM cells, while IL-6(10ng/ml) did not provide protection against ZD55-TRAIL-induced cell death, suggesting that ZD55-TRAIL, unlike Dex, is able to inhibit cytokine-induced MM cell growth.(5) ZD55-TRAIL, at a MOI of16for24h, significantly increased DNA fragmentation in RPMI8226cells, indicating that MM cells underwent apoptosis on ZD55-TRAIL infection.(6) ZD55-TRAIL at the indicated MOIs effectively induced cleavage of Caspase-8,-9,-3and PARP. The levels of TRAIL expression increased in a dose-dependent manner, while the expression levels of DR4and DR5were not affected.(7) The virus decreased the level of IGF-1R as well as NF-κB pathway proteins. Moreover, quantitative PCR analysis showed that the mRNA expression of IGF-1R was also significantly inhibited by ZD55-TRAIL in a time-dependent manner.(8) Exposure of MM cells to IGF-1(100ng/mL) could not reverse cellular inhibition and apoptosis induced by ZD55-TRAIL.Section2:The mechanism of inducing cell apoptosis with the combination of oncolytic virus ZD55-TRAILwith LY294002or MG132on myeloma cells.Aim:To investigate the mechanism of enhanced apoptosis induced by oncolytic virus ZD55-TRAIL with PI3K inhibitor LY294002or proteasome inhibitor MG132on MM cells. Methods:Cell growth was measured by MTT assay, Western blot method was used to analyze the expression of Caspase3, PARP, PI3K/Akt/mTOR downstream proteins. Colony forming assay was used to observe cells cloning ability, and the cytotoxicity of virus on normal liver cells LO2was assessed by crystal violet staining.Results:(1) LY294002synergized with ZD55-TRAIL to inhibit cell proliferation in myeloma cells.(2) blotting analysis showed that ZD55-TRAIL alone activated AKT and mTOR, while addition of the PI3K inhibitor LY294002led to increased levels of cleaved forms of caspase-3and PARP, and enhanced inhibition of key components of AKT/mTOR pathway was observed in the RPMI8226cells.(3) low-dose MG132potentiated low-dose ZD55-TRAIL-mediated cell death in RPMI8226cells, with the combination index＜1. Moreover, they did not induce cytotoxicity against normal cells LO2.(4) Western blotting showed an enhanced caspase cascade activation in the cells treated with the two agents, which was related, at least in part, to upregulation of DR5.(5) ZD55-TRAIL and MG132reduced colony formation ability of MM cells in vitro.(6) MTT assay and crystal violet staining showed ZD55-TRAIL, MG132, or combined treatments did not induce cytotoxicity in normal cells.Conclusion:ZD55-TRAIL induces significant cytotoxicity in drug-sensitive and-resistant myeloma cell in vitro. Furthermore, we show for the first time that ZD55-TRAIL markedly inhibits the expression of IGF-1R and downstream NF-κB. Combination of ZD55-TRAIL with LY294002in RPMI8226cells overcomes the activation of mTOR and AKT induced by the virus and consequently promotes cell death. Moreover, MG132upregulated DR5and potentiated ZD55-TRAIL-mediated cell death in RPMI8226cells.