Study Of Cancer Treatment Using Reconstituted High Density Lipoprotein-Doxorubicin

Our research aimed to explore the possibility of using rHDL in delivering chemotherapeutic agents to tumor and overcoming cancer drug resistance. We also studied the role of autophagy in cancer drug resistance.In the fisrt part, we prepared rHDL/Dox; observed the drug accumulation effect of rHDL/Dox using confocal observation and FACS analysis; evaluated the cytotoxicity of rHDL/Dox using MTT and western blot; tested the liver targeting effect using vivo imaging. In the second part, we showed the drug resistance of KBV cells using real time PCR and FACS analysis; observed the effect of overcoming drug resistance using MTT, western blot, apoptosis detection, tunel observation. In the third part, we showed that rHDL/Dox induced autophagy by western blot, Cyto-ID staining, EGFP-LC3 transfection and transmission electron microscopy; then we showed the role of autophagy in cancer drug resistance through using autophagy inhibitors and siRNA technology.rHDL/Dox showed good size distribution and stability in vitro. Through the interaction of SR-B1 receptor, rHDL/Dox could accumulate more drugs intracellular in these two cell lines; induce more significant caspase-3 dependent apoptosis and the release of LDH in HepG2 and SMMC-7721 cells; deliver drug effectively to liver in vivo. Drug resistant KBV cell showed a relative high expression of SR-B1 receptor. Compared with free doxorubicin and Lipodox, rHDL/Dox could reverse the pump effect of P-glycoprotein; accumulate more drugs intracellular; induce more significant caspase-3 dependent apoptosis and the cleavage of DNA. We also showed that treating KBV with rHDL/Dox could induce cell autophagy and autophagy strengthened the tolerance and resistance of KBV cell to doxorubicin. Combining with autophagy inhibitors, chloroquine and LY294002, could help rHDL/Dox to reverse drug resistance and induce more significant cell apoptosis. Knockdown of autophagy related genes, Atg5 and beclin1, could also weaken the tolerance of KBV to rHDL/Dox. We further demonstrated that autophagy induced by rHDL/Dox was regulated by Akt/TOR pathway.In conclusion, rHDL/Dox could enhance the killing effect of doxorubicin to hepatocellular carcinoma cells. rHDL/Dox could overcome drug resistance through evading drug efflux. Suppression of autophagy helped rHDL/Dox overcome drug resistance.