Combination Of TRAIL-Armed ER Nanosomes With The Antagonist Of IAPs(AZD5582) As A Targeted Cancer Therapy

TRAIL(tumor necrosis factor related apoptosis inducing ligand)is a tumor necrosis factor,which can specifically induce cancer cells apoptosis,but no effect on normal cells.Therefore,TRAIL has a good application prospect in cancer treatment.However,studies have shown that due to the short half-life,bioavailability of rttrail and the drug resistance of cancer to trail,the overall effect of r TRAIL is not ideal,only for a small number of cancer patients.In order to overcome these shortcomings of TRAIL,this research prepared an endoplasmic reticulum(ER)-derived nanosomes TRAIL(ERN-T)for cancer treatment.The specific research contents and results are as follows:(1)Endoplasmic reticulum nanocarriers(ERN-T)were prepared from the endoplasmic reticulum of MSCs expressing TRAIL by gradient centrifugation.The results showed that:1)When the TRAIL lentiviral vector was 30μL(MOI=3),more than 95%MSC expressed TRAIL.Immunofluorescence staining showed that TRAIL was highly expressed in the endoplasmic reticulum of MSC.2)3.1×10~9 ERN-T nanoparticles were isolated from 1×10~6MSCfl T cells by density gradient centrifugation.The size and morphology of the nanoparticles were analyzed by NTA and TEM.The size of the nanoparticles ranged from70.6 nm to 225.4 nm,showing a non-uniform monolayer vesicle structure.3)3.1×10~9ERN-T(33.4μg total protein)could be used for 2.2 ng TRAIL delivery;4)immunotransmission electron microscopy and Western blotting also showed that TRAIL was highly expressed on ERN-T.In a word,these results indicate that ERN-T is successfully used in TRAIL delivery.(2)Effects of ERN(without TRAIL),ERN-T,EV-T(extracellular vesicle carrying TRAIL)and r TRAIL on the proliferation of trail sensitive(H727),TRAIL resistant(M231)tumor cells and normal cells(Ha Ca T)in vitro show that the killing effect of ERN-T and EV-T was significantly stronger than that of r TRAIL not only on H727 but on M231.And the killing effect of ERN-T on H727 was significantly stronger than that of EV-T.The good killing effect of ERN-T on tumor is attributed to the TRAIL carried by ERN,because ERN has no effect on cells.More importantly,drugs could not inhibit the activity of Ha Ca T cells,indicating that ERN-T could selectively induce apoptosis of tumor cells.In order to further enhance the killing effect of ERN-T on cells with high TRAIL resistance,ERN-T and r TRAIL were evaluated with AZD5582.The results showed that more than 70%of the tumor cell activity was inhibited by 4.0 ng/m L ERN-T combined with 20 n M AZD5582(especially in M231 cells,more than 90%of the viability was inhibited),but the combination had no effect on A549,normal MSc and Ha Ca T.It was found that AZD 5582 could down regulate the expression of c IAP1,XIAP,Mcl-1,Survivin and other anti apoptotic proteins,thus sensitizing cancer cells to the apoptotic signal of TRAIL and significantly killing cells.In M231 subcutaneous xenograft tumor model,ERN-T has good tumor targeting.Intratumoral injection of PBS,ERN-T,AZD5582,r TRAIL combined with AZD5582 and ERN-T combined with AZD5582 were used to study their antitumor effects.The results showed that the inhibition effect of ERN-T combined with AZD5582 on tumor was not only significantly stronger than that of ERN-T and AZD5582 alone,but also significantly stronger than that of r TRAIL combined with AZD5582.H&E staining and immunohistochemical analysis showed that the combination of ERN-T and AZD5582 significantly inhibited the expression of Ki-67,thereby inhibiting the proliferation of M231 tumor.And the expression levels of Caspase-3 and TUNEL increased,indicating that AZD5582 significantly enhanced the apoptosis inducing effect of ERN-T on M231 tumor.In addition,the results of mouse weight and organ immunohistochemistry showed that the combination had no significant toxic and side effects on mice.To sum up,in this paper,we studied in detail the anti-tumor effect of a kind of endoplasmic reticulum nano bilayer membrane vesicle carrying TRAIL(ERN-T)combined with chemical AZD5582 on breast cancer M231,which showed good anti-cancer activity in vivo and in vitro,and analyzed its mechanism to provide an efficient and safe treatment strategy for cancer treatment.