The Anti-colon Cancer Effect And Mechanism Of Rapamycin Liposome

Rapamycin(Rapa)is a novel macrolide immunosuppressant.It has been reported that Rapa has cytotoxic effect on colon cancer,bladder cancer and cervical cancer.However,due to the defects of the poor water solubility and low bioavailability of Rapa,its clinical antitumor application is limited.The problem of water solubility was improved by the derivative of Rapa,everolimus.However,it is only used for second-line treatment of advanced renal and pancreatic cancer.Therefore,we speculate that improving the water-solubility of Rapa without increasing its accumulation in tumor sites in vivo will not effectively improve its antitumor effect,so how to effectively deliver the drug to the tumor sites is a new treatment method.Based on our previous research,we selected Liposomes to encapsulate Rapa(Rapamycin Liposomes,Rapa LPs)and Rapa LPs were prepared.In vitro cytotoxicity,in vivo distribution and the therapeutic effect of Rapa LPs on colon cancer were investigated in cell and animal experiments.5-fluorouracil(5-FU)is first-line chemotherapeutic agents used for the treatment of colon cancer.However,5-FU has toxicity to normal cells and acquired resistance of tumor cells.Therefore,it was further investigated whether the combination of rapamycin liposome and 5-fu could improve the treatment effect of colon cancer.The main research contents of this paper are as follows:Chapter 1: Preparation and characterization of Rapa liposomesObjective: To prepare Rapa nanostructured lipid carriers.Methods: Rapa LPs were prepared via ethanol injection method.The particle size and zeta potential of Rapa LPs were measured by the laser particle size analyzer analyzes.The shape was observed using transmission electron microscope(TEM).The amount of Rapa in liposomes was quantified by HPLC method.Cell uptake experiment observe the uptake of the drug by the cells.The cytotoxicity of Rapa LPs and Rapa was analyzed by MTT method.The in vitro cytotoxic activity of Rapa LPs was determined using MTT assay in colon cancer cells.Result: The Rapa LPs had a very narrow particle size distribution(PDI=0.125-0.25)with a mean particle size of 100 ± 5.5 nm.The zeta potential of Rapa LPs was close to neutral.TEM shown that Rapa LPs were spherical in shape and similar DLS measurement results.The entrapment efficiency and drug loading of Rapa LPs is80 ± 2.5 % and 9 ± 0.5 %,respectively.The drug intake of the Rapa LPs group was higher than that of the free Rapa.In addition,MTT assay showed after Rapa LPs was applied to HCT-116 cells and SW480 cells for 48 h,50% concentration of inhibition of HCT-116 cells and SW480 cells was 5.61 ± 0.75 μg/mL,7.52 ± 0.51 μg/mL,but of free Rapa was 15.34 ± 1.18 μg/mL,12.22 ± 1.08 μg/mL.Conclusions: Rapa LPs were prepared with high encapsulation efficiency and good stability.Rapa LPs promotes drug uptake and is more cytotoxic to colon cancer than Rapa API at the same concentration.Chapter 2: The antitumor effect of rapamycin liposomes in vivo.Objective: To investigate the distribution of Rapa LPs in HCT116 tumor-bearing mouse and to study the efficacy of the combination of Rapa liposome and 5-FU in HCT-116 tumor-bearing mice.Methods: HCT-116 tumor-bearing mice were used to assay the therapeutic efficacy of Rapa LPs.DIR liposomes were prepared and injected into the tail vein in order to observe the fluorescence distribution in mice.The anti-colon cancer efficacy of free Rapa,Rapa LPs,5-FU and Rapa or Rapa LPs combined with 5-FU group were investigated in HCT-116 tumor-bearing mice.Ki67 immunohistochemistry and TUNEL staining were used to detect tumor cell proliferation and apoptosis,respectively.Result: Compared with free DIR,DIR liposome have strong fluorescence signal in inoculated tumor.The tumor growth curves are proved that the anti-tumor efficacy of Rapa LPs was superior to free Rapa,and the tumor inhibition rate of Rapa LPs combined with 5-FU group was 89.8 %(P<0.05),which was significantly higher than the Rapa LPs group(57 %)(P<0.001)and the 5-FU group(53 %).The cell proliferation inhibition rate and apoptosis rate of the Rapa LPs group were significantly higher than free Rapa group.Moreover,the Rapa LPs combined with5-FU group were higher than the Rapa LPs or 5-FU single drug treatment group(P<0.01).In addition,Rapa LPs combined with 5-FU also inhibited the expression of the main proteins related to epithelial-mesenchymal transitions.The drug has no obvious toxicity to the organs and tissues of nude mice.Conclusions: The Liposomes enhancing drug delivery to tumors,Rapa LPs treatment inhibited colon cancer cell growth more effectively than the free Rapa treatments,while the Rapa LPs combined with the 5-FU group was significantly better than the Rapa LPs or 5-FU single drug treatment group,which had a synergistic efficacy on colon cancer.Chapter 3: The anti-tumor effect of rapamycin liposomes in vitro.Objective: To study the effects of rapamycin liposomes,5-FU and the combination of these two drugs on the proliferation,apoptosis,invasion and migration of human colorectal HCT-116 and SW480 cells in vitro.Methods: MTT was used to observe the antitumor effect of Rapa LPs combined with 5-FU on HCT-116 and SW480 cells.Flow apoptosis kit was used to detect the apoptosis of tumor cells.Finally,Transwell assay was used to detect the migration of tumor cells.Result: Compared with free Rapa,Rapa LPs could better inhibit the proliferation,invasion and migration of HCT-116 and SW480 cells,and induce cell apoptosis.Moreover,the combined treatment of Rapa LPs and 5-FU was better than that of Rapa LPs or 5-FU monotherapy.Rapa LPs and 5-FU inhibit cell invasion and migration by inhibiting related proteins of epithelial interstitial transformation.Conclusions: The anti-tumor effect of Rapa LPs group was better than that of free Rapa,which further confirmed the effect of Rapa LPs and 5-FU in the treatment of colorectal cancer.Chapter 4: Study of the mechanism of rapamycin liposomes on colon cancer in vitro.Objective: To study of the mechanism of Rapa LPs,5-FU and the combination of the two drugs in vitro.Methods: Western blot was used to detect the expression of related proteins.The effects of Rapa LPs combined with 5-FU on the mitochondrial pressure and glycolysis of hct-116 cells were observed by cell energy metabolism analysis.The content of lipid droplets in the cells was detected by oil red O staining.Results: Rapa LPs synergized the anti-tumor effect of 5-FU by inhibiting the AKT/mTOR pathway and activating the P53 pathway to up-regulate the protein expression of Bax,they promoted the release of cytochrome C and the activation of caspase pathway,which eventually led to the endogenous apoptosis of HCT-116 and SW480 cells and played a synergistic anti-tumor role in vitro.Compared with free Rapa,Rapa LPs can better inhibit the aerobic phosphorylation of mitochondria and glycolysis of HCT116 cells,and causes the accumulation of lipid droplets within the cell.The combined treatment of Rapa LPs and 5-fu was better than that of Rapa LPs or 5-FU monotherapy.Conclusion: Rapa LPs and 5-FU play an anti-colon cancer role by inhibiting AKT/mTOR pathway and activating P53 pathway and interfering with aerobic phosphorylation,glycolysis and lipid metabolism of tumor cells.In summary,cellular uptake,in vivo imaging,and cell uptake experiments,it was found that liposome group enriched more in tumor than those in the Rapa LPs group.Therefore,The results in vitro and in vivo showed that the inhibition of tumor proliferation,migration and apoptosis in the Rapa LPs group was superior to that in the free Rapa.In addition,the effect of Rapa LPs combined with 5-FU in the treatment of colorectal cancer was also better than that of the single-drug treatment group,and it did not increase the toxicity to the visceral tissues,which showed a synergistic anti-tumor effect.Further mechanism study demonstrated that Rapa LPs synergized the anti-tumor effect of 5-FU by inhibiting the AKT/mTOR pathway,activating the P53 pathway,and interfering with tumor aerobic glycolysis and lipid metabolism.In general,rapamycin delivered by liposome not only improved the anti-tumor effect of Rapa,but also synergistically improved the anti-tumor effect of5-FU.