Study On The Effect And Mechanism Of Cationic Polysaccharide Spermine-pullulan In Tumor Treatment

Objective:Polysaccharide derived from animal,plant and microorganisms possessing various advantages,such as good biocompatibility and easy to modify,various kinds of cationic polysaccharide can be obtained by introducing positively charged group.A growing number of studies have revealed that cationic polysaccharide can be applied in antitumor research as good vehicle for the delivery of nucleic acids,protein/polypeptide and chemotherapeutic drugs,and several cationic polysaccharide itself were found immunostimulatory effect,which can promote the mature of dendritic cells or affectt the phenotype of macrophages.Spermine modification imparts positive charge to pullulan,forming the cationic polysaccharide pullulan-spermine(PS),PS as gene delivery carrier for hepatocyte,endothelial cell and neurocyte exhibited unique advantages among cationic polysaccharide for its high affinity to asialoglycoprotein receptor.In the previous studies,we observed that PS could mediate effective RNA interference(RNAi)in hard-to-transfect leukemia cells by targeting gene which associated with drug-resistance.Malignant tumor can be divided into solid tumor and hematological tumor.In solid tumor,the immunosuppressive M2 phenotype tumor-associated macrophage(TAM)is an important target in anti-tumor immune therapy,while the mutated gene is an excellent therapeutic target in hematological malignancy.Based on the previous works and the related research background,the aim of this project was to synthesize cationic polysaccharide PS,and systematic research on its effect on macrophage and the relative molecular mechanism,followed by its application in breast cancer mice model,and PS was also applied as siRNA delivery carrier to investigate the BCR-ABL gene silencing function in chronic myeloid leukemia(CML)cells.Methods:The murine macrophage Raw 264.7 was served as MO phenotype,and incubated with lipopolysaccharide(LPS)and IFN-γ or IL-4 to induce MO macrophages polarizing toward M1 or M2 phenotype,respectively.CCK-8 assay was used to determine the cytotoxicity effect of PS or PS combined with LPS(PS+LPS)on macrophage and the effect of PS-siRNA on three kinds of CML cells K562、MEG-01 and Ku812.The uptake of PS or PS+LPS by macrophages and PS-siRNA by K562,MEG-01 and Ku812 cells were examined by flow cytometry.The localization of PS in macrophage was observed by confocal imaging.The effect of PS or PS+LPS on macrophage phenotype markers(TNF-a,IL-1β,IL-6,IL-12,iNOS and CD206),antigen-presenting associated proteins(MHC-I,MHC-II and CD86)and Toll-like receptors(TLRs)were measured by Real-time PCR and flow cytometry.The activation of MyD88,MAPKs(Erk,JNK,P38),Akt,c-Jun and NF-κB were analyzed by western blot.The Real-time PCR and Western blot were conducted to measure the BCR-ABL gene silencing efficiency by PS in three kinds of CML cells,and the proliferation and differentiation of CML cells were examined by flow cytometry.In breast cancer models,the phenotype change of TAM in tumor tissue was determined by immunofluorescence staining and confocal imaging,the activation of T cells was tested by flow cytometry,enzyme-linked immunosorbent assay and cytotoxic immunological experiment,the metastasis in liver and lung were analyzed by histopathological examination,the expression of CD31 and MMP-9 were examined by immunofluorescence staining,and tumor growth and mice survival were monitored.Results:PS was highly taken up by MO or M2 macrophage and located in lysosome.PS promoted the expression of TNF-a,iNOS and inhibited CD206 in normal or IL-4 containing medium,and could also increase the expression of antigen presenting molecules CD86,MHC-I and MHC-II,which together promoted MO or M2 macrophages polarizing toward M1.The mechanism study of PS on macrophage revealed that PS upregulated the expression of TLR1,TLR3 and TLR4,promoted the phosphorylation of Akt,Erk,JNK,following the activation of NF-κB.In a murine breast cancer model,PS promoted the polarization of TAM towards M1,increased CD4~+and CD8~+T cells,which mediated stronger cytotoxicity to tumor cell.PS reduced the expression of MMP-9 and CD31 in the tumor,inhibited the angiogenesis in tumor and the metastasis to the lungs and liver,prolonged the mice survival.PS itself did not affect the expression of IL-1β,IL-6 and IL-12,but enhanced the immunostimulatory effect of LPS on macrophage,and promoted the expression of IL-1β,IL-6,IL-12,TNF-α and iNOS,improved the immunotherapeutic effect of LPS in breast cancer mouse model.PS mediated efficient transfection of BCR-ABL siRNA in three kinds of CML cells.The PS-BCR-ABL siRNA with N/P ratio of 2.5 was highly took up by three kinds of CML cells and the positive rate was up to 98%,and the RNAi efficiency peaked at 24 h,the silencing efficiency was 55%,38%and 29%under the condition of the 10%serum medium,and was 82%,46%and 62%in serum-free medium in K562,MEG-01 and Ku812 cells,respectively.PS-BCR-ABL siRNA significantly down-regulated the BCR-ABL protein,and inhibited the proliferation of three kinds of CML cells,induced the erythroid differentiation of K562 cells and the erythroid and megakaryocytic differentiation of MEG-01 cells.Conclusions:PS activated the signaling pathway mediated by TLR1,TLR3 and TLR4,promoted the antigen presentation ability of macrophage,and led to the polarization of macrophage towards M1.In murine breast cancer model,PS reprogrammed TAM to M1 phenotype,restored the tumoricidal ability of T cell,inhibited tumor growth and metastasis,and significantly prolonged the mice survival.PS combined with LPS showed synergistic therapeutic effects.PS mediated effective RNAi of BCR-ABL as gene transfection vector in CML cells,and inhibited cell proliferation,promoted cell differentiation.Thus,PS shows potential application prospects in gene function research and gene therapy in leukemia.