The Study Of Antitumor Effect Of Compound TRAIL Liposomes

Human recombinant TNF-related apoptosis-inducing ligand (TRAIL), also named as recombinant human Apo-2 ligand (Apo2-L), has an apparent molecular weight of 19493 Da by SDS-PAGE. Many reports have proven TRAIL to be a prominent biologically-targeted anti-tumor protein because of its remarkable ability to induce apoptosis in a variety of human cancer cell lines while leaving normal cells unaffected. Many primary tumors, however, are highly resistant to TRAIL, suggesting that the use of TRAIL alone may be ineffective against cancers. To restore the TRAIL-induced apoptotic potency, combination therapies of TRAIL with chemotherapeutic drugs have been studied, indicating that combination therapies may be an effective strategy for the treatment of tumors.In spite of modest progress, the clinical use of free chemotherapeutic drugs and TRAIL are limited by the follow factors.①TRAIL receptors are widely expressed on the surface of somatic cells and half-lives of TRAIL were only 3-5 min in rodents and 24-31 min in non-human primates, which may significantly reduce the efficacy of TRAIL. The pharmacokinetic study showed that most of the TRAIL had been eliminated 5 hours after intravenous injection. Large amounts of TRAIL are thus required to obtain tumor growth suppression.②Therapeutic potential of chemotherapeutic drugs has been limited by their side effects which may become more serious in the combination therapy with TRAIL.③The emergence of multidrug resistance (MDR) makes some tumor cell lines demonstrate cross-resistance to both TRAIL and chemotherapeutic drugs, making combination therapies ineffective. In this study, long-circulating liposomes were used as drug carriers to encapsulate TRAIL and chemotherapeutic drugs (doxorubicin and actinomycin D) with the aim to overcome above challenges.The first part described the construction and characterization of TRAIL liposomes (TRAIL-LP) and doxorubicin liposomes (DOX-LP). The DOX-LP were prepared according to the method using an ammonium sulfate gradient. The particle size and loading efficiency of DOX-LP were 98.4±19.2 nm and 96.2±3.6%, respectively. The sonication procedure under ice bath revealed slight influence on TRAIL bioactivity. In contrast, the freeze-thaw process demonstrated serious damage on the TRAIL biaoactivity. So TRAIL-LP were prepared according to the film dispersion technique, its particle size was decreased by sonication procedure. Accordingly, The particle size and loading efficiency of TRAIL-LP were 115.6±25.4 nm和10.4±3.9%. respectively. The dissolution study of liposomal drugs was also carried out in this report. The in vitro cytotoxic research showed that combined treatment with TRAIL-LP and DOX-LP resulted in much more cell inhibition than monotherapy.The second part investigated the passtive tumor-targeting property of the liposomes and tissue distribution of DOX-LP. The passive tumor-targeting property was determined non-invasively in A-549 tumor-or intracranial U87 tumor-bearing nude mice, based on the fluorescence of DiR. The results suggested that the more liposomal Dir accumulated in both A-549 tumor and U87 tumor than free Dir, demonstrating the tumor-targeting property of the liposomes. The biodistribution study indicated that thhere were significantly difference in tumor tissue between DOX-LP and free doxorubicin (p< 0.05). Importantly, the TRAIL-LP showed no obvious effect on the biodistribution of DOX-LP, suggesting that both DOX-LP and TRAIL-LP might be accumulated in the tumor tissue to enhance the antitumor effect.The third part showed the in vivo antitumor effect of TRAIL-LP and DOX-LP. The synergistic effect of combination treatment was reflected in the median survival time of mice bearing intracranial U87 tumor xenografts where the median survival time of mice treated with combination therapy was significantly longer than that of mice treated with DOX-LP or TRAIL-LP alone. In contrast, no prolonged median survival time of mice treated with free DOX and TRAIL combination was observed compared to mice treated with free DOX alone or TRAIL alone. Similarly, synchronously systemic administration of DOX-LP and TRAIL-LP also caused significant suppression of tumor growth of human A-549 xenografts in a synergistic fashion.The forth part presented the underlying mechanisms by which this combined treatment achieves enhanced killing of A-549 and U87 cells. Sensitization to TRAIL-LP mediated by DOX-LP was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5). The use of blocking agents revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. In addition, western blot analysis indicated that TRAIL-LP-mediated proteolytic processing of procaspase-3,-8,-9 was partially blocked in A-549 or U87 cells, treatment with DOX-LP efficiently recovered TRAIL-LP-induced activation of caspases. As anti-apoptotic proteins for the mitochondrial pathway, Mcl-1, Bcl-2 and Bcl-x expression level werelowered in A-549 cell line when they were treated with the combination of TRAIL-LP and DOX-LP. While the apoptotic protein of the Bcl-2 family, Bakl/2 seemed to be increased in A-549 cells after combined treatment. These results, taken together, indicated that both caspase activation and Bcl-2 family member are both play an important role in the combined therapy of liposomal drugs.In order to confirm the wide effectiveness of enhanced antitumor effect of TRAIL-LP and chemotherapeutic liposomes. The last part investigated the antitumor effect and mechanisms of TRAIL-LP and actinomycin D liposomes (ActD-LP). Our result showed that TRAIL-LP and ActD-LP can enhance antitumor effect on A-549 tumor bearing mice, suggesting that the combination of TRAIL-LP and chemotherapeutic liposomes has a good potential for cancer therapy.