Study On Apoptosis Of Tumor Cells Induced By Nucleolin Cross-linking On Cell Surface

The morbidity and mortality of malignant tumors（cancers）are still high due to the characteristics of rapid growth and easy metastasis,which has become a major public health problem that seriously threatens human health.Traditional treatment methods like surgery,radiotherapy,and chemotherapy are often limited by the poor targeting,side effects,incomplete tumor tissue resection,and high recurrence rate.Therefore,it is of great significance to develop new cancer treatment methods that can effectively kill tumor cells with low cytotoxicity to normal cells.In recent years,the aggregation of cell membrane receptors mediated by external factors can activate intracellular apoptosis signaling pathways,and trigger tumor cell apoptosis,which has become a novel and potent method of cancer treatment.However,the cell membrane receptors currently studied are very limited,and most of the therapy model is only useful for a certain kind of disease.It is difficult to achieve broad-spectrum anti-tumor therapy.In addition,the biological probes on the cell surface usually suffer from the problems such as short action time,poor stability,and easy endocytosis.Therefore,a novel strategy was developed to induce cancer cell apoptosis based on the cross-linking of nucleolin overexpressed on the surface of tumor cells to achieve anti-tumor therapy in vivo and in vitro.A multifunctional probe was constructed for long-term cell membrane anchoring,restricting the relative movement of the receptor and inducing the receptor to form a cross-linked state.In this case,real-time monitoring of membrane protein,inducing tumor cell apoptosis and anti-tumor therapy can be achieved in our work.It mainly includes the following two aspects of work:（1）Preparation of GC-chol-apt-cDNA and long-term cell membrane imaging:In this chapter,ethylene glycol chitosan（GC）was used as the polymer backbone,and Cy3dye-labeled oligonucleotides aptamer（Cy3-aptamer）and cholesterol were branched on its side through amide reaction to form the intermediate product GC-chol-apt.In order to reduce the false positive signal caused by the non-specific adsorption of the probe on the cell membrane,BHQ2-labeled cDNA（BHQ2-cDNA）was introduced and partially hybridized with aptamer to form a double-stranded structure,constructing a flexible"multi-catcher"complex GC-chol-apt-cDNA.Various techniques such as fluorescence spectra,gel electrophoresis,dynamic light scattering and atomic force microscopy have shown that GC-chol-apt-cDNA has been successfully synthesized,with a spherical structure of about 500 nm.The fluorescence co-localization experiment of cell membrane dye and nucleolin antibody showed that the probe can specifically target nucleolin on the surface of tumor cells,resulting to the release of cDNA and fluorescence recovery of Cy3,achieving accurate and specific targeted imaging of nucleolin on the surface of tumor cells.Furthermore,the structure was anchored to the cell membrane through the affinity of aptamer and the hydrophobic effect of cholesterol,which effectively inhibited endocytosis and can stably exist on the cell surface for a long time.The results of flow cytometry and fluorescence imaging experiments indicated that the affinity of GC-chol-apt to cells was increased by nearly 100 times comparing to the oligonucleotides aptamer,and it can stably anchor on the cell membrane for up to 6 hours,providing a useful tool for long-time cell membrane imaging.（2）Nucleolin cross-linking induces tumor cell apoptosis in vivo and in vitro and analysis of apoptotic pathway:In the multifunctional probe,aptamers branched on the"multi-catcher"GC-chol-apt-cDNA were used to capture the nucleolin in adjacent parts,while the polymer backbone of GC can make the distance close to form a cross-linked state.Furthermore,the binding of GC-chol-apt-cDNA on the cell membrane can stable for a long time through the multi-site membrane anchoring of cholesterol.Therefore,GC-chol-apt-cDNA can stably induce the formation of cross-linked state of nucleolin on the surface of tumor cells,showing obvious killing effect on a variety of tumor cells,but not normal cells.We further confirmed that GC-chol-apt-cDNA induced apoptosis of tumor cells in a dose and time-dependent manner at the cellular level through double staining of living and dead cells,quantitative analysis of apoptosis by flow cytometry and nuclear fluorescence imaging.The results showed that when the concentration was 100μg/m L,the cell viability rate was only 21%.In addition,after 8 hours of binding with the probe,the cell nucleus shrank and ruptured,and the complete cell nuclear structure was destroyed,indicating that the probe can effectively kill tumor cells and achieve anti-tumor therapy at the cellular level.Through the mouse tumor model,we further evaluated the anti-tumor activity of GC-chol-apt-cDNA in live animals.Through fluorescence imaging in vivo,isolated tumor tissue immunohistochemical staining and tumor growth curve analysis,we verified that GC-chol-apt-cDNA can effectively target the tumor site and induce cell apoptosis compared with the control group,which has excellent proliferation activity with 81%inhibition rate.Finally,immunofluorescence imaging and western blotting technology indicated that the increasing of intracellular Ca²⁺influx mediated by nucleolin cross-linking can cause the loss of mitochondrial membrane potential,and further release cytochrome C and downstream casapse enzymes that execute the apoptotic process.Therefore,we confirmed that GC-chol-apt-cDNA can activate the apoptotic signaling pathway through the mitochondrial pathway,and has been successfully applied to animal tumor solid models.In summary,the"multi-catcher"probe GC-chol-apt-cDNA can effectively target tumor cells,achieve long-term membrane protein imaging,activate intracellular mitochondria apoptosis signals by regulating nucleolin cross-linking,and inhibit the proliferation of tumor cells in vivo and in vitro ultimately.As a new anti-tumor method,GC-chol-apt-cDNA provides new ideas for inducing cell apoptosis,which offers the following attractive features:（1）firstly,the strategy of nucleolin cross-linking to induce tumor cell apoptosis is proposed for the first time to realize anti-tumor therapy.We combine the spatial regulation of nucleolin on the cell membrane with the intracellular apoptosis signal cascade,breaking through the conventional model of nucleolin as a drug target and carrier in anti-tumor therapy;（2）secondly,this probe has the advantages of simple synthesis,long-lasting action,strong selectivity,and is expected to become a new and universal anti-tumor drug for the treatment of malignant tumors.