Nucleolin Interference And Telomere Dysfuction In The Growth Inhibiton Of Glioma And Related Mechanisms

Glioblastoma(GBM)is one of the most common intracranial tumors,which is characterized by high degree of malignancy and poor prognosis.Surgical treatment of GBM is limited by a high recurrence risk.Certain drug resistance is also occurred in chemotherapy.Nucleolins are commonly found in eukaryotic cells,regulating ribosome aggregation,DNA metabolism and RNA expression and other biological activities.The transcriptional characterization of nucleolus-regulated m RNA is the primary role in controlling cellular biological activity.Nucleolin consists of 707 amino acid residues,including three main functional domains: mainly carboxyl terminal,amino terminal and central region.Amino acid terminal regulates c DNA transcription,ribosome precursor packaging.Central region contains four conservative,consistent RNA binding domains(RBD).Carboxyl terminal contains rich glycine residues,through which RNA can be more easily accessible to the central area of RBD structure.The RBD of nucleolin has the ability to bind to a series of m RNAs specifically,including Bcl-2,p53 and other genes that regulate cell apoptosis.AS1411 is a 26-mer-rich nucleic acid ligand that is currently used in clinical phase II trials for tumor therapy.It has a G-rich sequence,which has the advantage of being easily absorbed by ribozyme degradation and therefore has the potential to be used to tumor treatment.It has been reported that AS1411 can competitively bind to RBD region of nucleolin and therefore has the ability to interfere with numorous nucleolus regulatory proteins.Commonly known as arsenic,arsenic trioxide(As2O3)is an traditional Chinese drug.In antient times,Chinese people use As2O3 and other arsenic compound to treat a variety of stubborn diseases such as asthma.In recent years,As2O3 has been gradually applied to the treatment of cancer.As2O3 can inhibit telomerase and thus induce biological effects on cells.So far,nucleolin interference and telomerase inhibition in the glioma has not heen wellreported.Because these two proteins are highly expressed in malignant glioma cells,we chose AS1411 to interference with nucleolus and use As2O3 to inhibit telomerase activity.And then explore the two drugs through the role of two key proteins to the tumor killing effect.Finally,because nucleolin can also regulate the expression of telomerase,we further explore the telomerase inhibition phenomenon induced by AS1411 via nucleolin.In this paper,we use glioma cell lines,glioma tissue,normal brain tissue,astrocytes and xenograft models as the experimental subjects.We use MTT,Telomeric repeat amplification protocol(TRAP)assay,immunofluorescence,western blotting,chromatin immunoprecipitation(Ch IP),si RNA gene silencing,Real time-RT-PCR,flow cytometry,aging staining and telomeric fluorescence in situ hybridization analysis to investigate the interference of arsenic in AS1411 and the inhibition of telomerase by arsenic trioxide The corresponding cell apoptosis,cell cycle arrest,cell senescence and the main mechanism of these phenomena.Our main experimental results are as follows:(1)AS1411 can up-regulate p53 and down-regulate Bcl-2 and Akt1 by competitively binding RBD region of nucleolin.AS1411 inhibits the invasion and migration of glioma cells by down-regulation of Akt1 via nucleolin.The upregulation of p53 and down-regulation of Bcl-2 leads to early cell apoptosis.(2)Nucleolins are over-expressed in glioma tissue and glioma cells.AS1411 induces growth inhibition of glioma cells by interfering with nucleolin,while in vivo experiments,AS1411 significantly reduces tumor volume and prolong animal survival time.(3)As2O3 generates reactive oxygen species(ROS)in glioma cells.Resulting in telomerase nuclear to cytoplasm translocation and telomerase phosphorylation,which resultes in telomerase activity inhibition.(4)Inhibition of telomerase activity incdues DNA damage which occurs in telomere fraction of the chromosome then leads ATM and ATR based DNA damage.(5)As2O3 induces the dissociation of TRF2 and POT1(telomere binding protein)from telomere.Further,we find that As2O3 shortens telomere DNA 3suspension of telomere in glioma cells.(6)Telomere damage leads to apoptosis,cell cycle arrest and cell senescence in glioma cells(7)Nucleolin interference led to the inhibition of telomerase activity,which led to cell senescence and related phenomenons.Conclusion:In this paper,we first report that the up-regulation of p53 and down-regulation of Bcl-2 and Akt1 induced by AS1411 via nucleolin.We also demonstrate that inhibition of telomerase activity can cause glioma cell growth inhibition,apoptosis,cycle arrest and cell senescence.