The Study Of Anti-glioma Effects Of Compound TRAIL/DOX Liposomes

It is reported that brain glioma accounts for30-40%of all brain tumors. Despite the combined treatment reigimens including surgery、radio-and chemotherapy, the prognosis of brain glioma remains poor. So developing novel treatments is now becoming the key issue of brain glioma treatment.Apoptosis plays an important role in the process of embryogenesis and histodifferentiation. The imbalance of apoptosis is very important for tumorigenesis. Apoptosis can be executed through a mitochondria-dependent (intrinsic) and a death receptor-dependent, mitochondria-independent (extrinsic) pathway. The intrinsic pathway can be initiated by radiotherapy or chemotherapy while the extrinsic pathway can be initiated by ligand-induced activation of death receptors on the cell membrane. So in this study we used the chemotherapy agent Doxorubicin (DOX) and one of the members of tumor necrosis factor family, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as treatment agents, and employed the long-circulating liposome as the drug carrier, to formulate the novel compound liposomes, TRAIL-LP/DOX-LP. We hope our novel formulation could(?)combine the intrinsic and extrinsic pathway to induce the apoptosis of the tumor cells and at the same time take advantage of the enhanced permeability and retention effect (EPR effect) to increase the concentration of these two agents at the tumor site and at last induce the apoptosis of glioma.The first part described the study of synergetic anti-tumor effects of TRAIL combined with DOX. We chose human glioma U87cells which were not sensitive to TRAIL-induced apoptosis as the study objects.We firstly studied the in vitro synergistic anti-tumor effects of TRAIL and DOX, and then in vivo. The results showed that TRAIL and DOX had great synergistic effects in vitro while showed no significant anti-tumor effects in vivo. These results indicated that a novel drug delivery system may be needed. To vadidate the universality of the synergetic anti-tumor effects of TRAIL combined with DOX, we also studied about the the human non-small cell lung cancer in vitro and in vivo.The second part described the preparation and characterization of TRAIL liposome (TRAIL-LP) and doxorubicin liposome (DOX-LP). The DOX-LP was prepared according to the ammonium sulfate gradient method and TRAIL-LP was prepared according to the film dispersion technique and then we mixed the prepared DOX-LP and TRAIL-LP at different radio to formulate compound liposomes. The particle size of DOX-LP and TRAIL-LP were101.0±45.3nm and107.6±47.8nm, respectively, and the entrapment efficiency of them were respectively97.4%and12.5%. The compound liposome exhibited nearly spherical shape of moderate uniform particle size. Based on the fluorescence of DiR, we investigated the passive tumor targeting property of liposome. The results suggested that the liposome could accumulate at the tumor site.The third part studied the anti-glioma effects of the compound liposome TRAIL-LP/DOX-LP in vitro and in vivo, after that, the in vitro and in vivo safety property research were carried out.The in vitro cytotoxic research showed that combined treatment with TRAIL-LP and DOX-LP resulted in much more cell inhibition than monotherapy.The in vivo synergistic anti-glioma effects of TRAIL-LP/DOX-LP were studied through various experiments, including the tumor volume measurement, the expression of apoptosis inducing factor actived caspase-3, tumor cell apoptosis rate and the survival time of glioma-bearing mice administrated with different formulations. The results showed that, compared with the control group, TRAIL-LP group and DOX-LP group, the tumor volume of the compound liposome group was6.73±1.79mm3, which was significantly reduced (p<0.01) and the tumor cell apoptosis rate was also significantly increased, which was19.92±2.49%(p<0.02). The synergistic effect of the compound liposomes was also reflected in the median survival time of mice bearing U87glioma where the median survival time of mice treated with TRAIL-LP/DOX-LP was significantly longer than that mice treated with TRAIL-LP or DOX-LP alone. We also found that the expression of actived caspase-3which plays a very important role in apoptosis was increased. All the in vivo studies about anti-tumor effects indicated that the compound liposomes had great synergistic anti-glioma effects.The in vitro safety study employed the normal brain cell BCEC as the model. The results showed that there was no significant difference between the inhibition effects of TRAIL-LP/DOX-LP and DOX-LP, which indicated that the compound liposomes had good safety in vitro. On the other hand, the H&E staining method demonstrated the good safety property of TRAIL-LP/DOX-LP in vivo.The last part investigated the mechanisms of the synergistic effects of the compound liposomes TRAIL-LP/DOX-LP, in vitro and in vivo. The in vitro results showed that the sensitization to TRAIL-LP mediated by DOX-LP was accompanied by the up-regulation of death receptors (DR4) and5(DR5). In addition, the western blot analysis results indicated that the precursor forms of caspase-8, which played and important role in the extrinsic pathway and caspase-9, which played an important role in the intrinsic pathway had both decreased, while the active form of caspase-3had increased after the U87cells were treated with DOX-LP/TRAIL-LP. These results demonstrated that the synergistic anti-glioma effects of DOX-LP/TRAIL-LP were related to both the intrinsic and extrinsic pathway. We also demonstrated by immunofluorescence of the glioma sections that the DOX-LP could increase the expression of DR5in vivo.