Targeted Blockage Of STAT3 Signaling Pathway With Decoy ODN Suppresses Growth Of Human Glioma Cells

Human glioma is the most common primary tumor of human central nervous system.The high-grade gliomas,which are highly invasive and aggressive tumors, have been resistant to conventional treating modalities including surgical resection, radiotherapy and chemotherapy.Despite the great advances made in the treatments recently,the median survival for patients with high-grade gliomas is still less than one year.Therefore,it has always been one of focus of neurosurgery to explore the novel and effective treatment.At present,dued mainly to some oncogenes been simply intervened in the previous studies,the gene therapy research for glioma has not gained the expected major breakthrough.Carcinogenesis is a multiple-link,multi-stage,and multi-step process,which involves in a great number of oncogenes and antioncogenes. So it is difficult to cure glioma only by intervening one cancer-related gene.The novel treatment will not benefit until it can simultaneously modulate various cancer-related genes' expression.In recent years,it has been realized that some transcription factors, which is the key to modulate various cancer-related genes in the corresponding cell signaling pathway.The transcription factors can simultaneously and effectively modulate the transcription and translation of its downstream oncogenes,and it may be regarded as "general switch" that affect cancergenesis and aggravation.Through specific inhibition of the key transcription factor,a "cascade" or "waterfall"-like synthetic blockage to a variety of oncogenes will attain.Therefore it has gained great interests for target inhibition of transcription factor in studies of gene therapy for a variety of cancers,including human glioma and others.The transcription factor STAT3(signal transducer and activator of transcription 3) which controls the transcription activation of its downstream genes,including bcl-xl, bcl-2,c-myc,cyclinD1,survivin,mcl-1 and VEGF,is involved in the embyro development,physiologic regulation of cell growth,survival and differentiation,and pathogenesis of malignancies by participating in Jak2/STAT3 signaling pathway.Like many other malignancies,aberrant STAT3 activation was found in primary malignant gliomas and numerous cancer-derived cell lines.The constitutive activation of STAT3 contributes to the apoptosis inhibition and cell proliferation by up regulation of various oncogenes,which lead to the transformation and progression of glioma.So STAT3 has been defined as one of transcription factor-like oncogene.Experiments have showed that after STAT3 signaling pathway was significantly blocked,it could down-regulate the expression level of various oncogenes,and subsequently resulted in a depressed proliferation and increased apoptosis in human glioma cells.The result suggests that STAT3 acts as a promising molecular target of gene therapy for glioma.The decoy ODN strategy is based on the competiiion between the endogenous cis-element within the regulatory regions of target genes promoters and exogenously added molecules mimicking the specific cis-elements.Transfection of STAT3 decoy ODN,a small molecule DNA synthesized in vitro and with high compatibility,into cells will competitively inhibit the binding of STAT3 with its endogenous ciselements, prevent the transcription and activation of the oncogene,and subsequent result in cells apoptosis and inhibition of glioma cells proliferation.Because decoy ODN strategy is better than other approaches in the aspect of efficiency,cost,and specificity,it has been widely used as a novel and effective approach in a number of laboratory and clinical research for cancer gene therapy.Up to now,decoy ODN was adopted to inhibit STAT3 pathway in squamous cell carcinoma of the head and neck (SCCHN),human breast cancer and human lung cancer.The results have showed that STAT3 signaling pathway could be significantly blocked,which subsequently resulted in the expression level downregulation of some oncogenes,including cyclinD1,c-myc, survivin,VEGF,bcl-2 and bcl-xl,and the depressed proliferation and increased apoptosis in the three cancer cells.It illuminates the study of therapeutic potential of STAT3 decoy ODN to human gliomas.In the current study,decoy ODN targeted inhibiting STAT3 pathway was employed to investigate the influence to human glioma cells.The study was divided into three parts.Partâ… Overexpression and Constitutive Activation of STAT3 in Human Glioma Samples and Cell LinesObjectiveTo study the expression level of STAT3 and its phosphorylation protein in human glioma cell lines and clinical specimens,and the significance of overexpression and constitutively activation of STAT3 signaling pathway in the pathogenesis of human glioma.Methods1.STAT3 and phosphorylated-STAT3 protein expression of 24 cases of human glioma samples were detected by Western blot analysis,and phosphorylated-STAT3's frequence was evaluated in human glioma.2.The protein expression of human glioma cell lines U251 and A172 were detedted by Western blot analysis with anit-STAT3 and anti-phospho-specific STAT3 (Tyr705,Ser727)antibody.Results1.STAT3 were expressed at higher level in the human malignant glioma samples than normal neural tissues.Furthermore,in the human malignant glioma samples, 75%(18 of 24 cases)expressed Tyr-705-phospho-STAT3 and 66.67%(16 of 24 cases) expressed Ser-727-phospho-STAT3.In contrast,there was no present in nomal neural tissue.2.Overexpressed STAT3 and its two active forms,Tyr-705-phospho-STAT3 and Ser-727-phospho-STAT3,were identified in both U251 and A 172 cells.Conclusion1.Overexpression and constitutively activation of STAT3 are widely presented in human glioma samples and cell lines,it suggests that aberrant STAT3 signaling pathway may play an important role in the pathogenesis of human glioma.2.Overexpression and constitutively activation of STAT3 are presented in U251 and A172,so the two cell lines could be used as ideal models in our in vitro study.Partâ…¡Targeted Blockage of STAT3 Signaling Pathway with Decoy ODN Suppresses Growth of Human Glioma CellsObjectiveTo study the the transfection efficiency of STAT3 decoy ODN,the specificity of decoy ODN to STAT3 signaling pathway,and the inhibiting potency of STAT3 decoy ODN in glioma cell proliferation.Methods1.STAT3 decoy ODN was transfected into U251 and A172 cells as mediated by Lipofectamine^TM2000.2.Flow cytometry was used to examine the transfection efficiency of STAT3 decoy ODN in U251 andA172 cells.3.Fluorescence microscope was used to observe whether STAT3 decoy ODN was transfected into U251 and A172 cells.4.Luciferase reporter assay was used to examine the specificity of decoy ODN to STAT3 signaling pathway.5.Cell counting assay was used to examine the inhibitory effect of STAT3 decoy ODN in glioma cells.6.MTT assay was used to analyze the proliferation inhibiting effects in U251 and A 172.7.[³H]-thymidine incorporation assay was performed to detect the inhibiting consequence of two cell lines DNA synthesizing.Results1.STAT3 decoy ODN could be highly efficiently transfected into glioma cells as mediated by Lipofectamine^TM2000.The transfection efficiency detected with flow cytometry showed that the decoy ODN was introducted into U251 and A172 cells efficiently in a dose-dependent manner.The MFI means of U251 and A172 cells reached a maximum of 64.03%and 56.22%at 50 nmol/L decoy ODN,respectively. Moreover,according to the viewing by fluorescence microscope,the decoy ODN had been incorporated into glioma cells,partly in the nuclei.2.Decoy ODN specifically blocked STAT3 signaling pathway in glioma cells. According to STAT3-1uciferase reporter assay,STAT3 decoy ODN substantially reduced transcription activity of STAT3 in U251 and A172 cells(P＜0.05).However,it didn't cause similar decrease in STAT6 transcription activity.Additionally,by western blot analysis,we found that STAT3 decoy ODN did not have significant effect on the level of phosphorylated STAT3(Tyr705 and Ser727)in U251 and A172 cells.3.STAT3 decoy ODN could effectively inhibit cell proliferation in human glioma.The result of cell counting assay demonstrated that the growth of both U251 and A172 cells were inhibited significantly after treatment with STAT3 decoy ODN and exhibited a time- and dose-dependent manner(P＜0.05).Consistent with the observation,the inhibition rate detected by MTT assay also confirmed that STAT3 decoy ODN contributed to potent growth inhibition in the two cell lines in a time-and dose-dependent manner(P＜0.05).[³H]-TdR incorporation assay revealed that CPM gradually reduced for the increase of STAT3 decoy ODN.Compared with the control group(TE),it was significant for DNA synthesis inhibition dued to 25 and 50 nmol/L STAT3 decoy ODN(P＜0.05).Conclusion1.STAT3 decoy ODN can be highly efficiently transfected into glioma cells as mediated by Lipofectamine^TM2000.2.Decoy ODN can specifically block STAT3 signaling pathway by inhibiting its transcription activation.3.STAT3 decoy ODN can contribute to the growth suppression in human glioma cells with little side effect.Partâ…¢Relevant Mechanisms of Proliferative Inhibition via Targeted Blocking STAT3 Signaing Pathway with Decoy ODN in Human Glioma Cells ObjectiveTo study the relevant mechanisms on proliferative inhibition of targeted blocking STAT3 signaling pathway by the decoy ODN in the glioma cell lines.Methods1.Flow cytometry was used to examine the change of cell cycle after STAT3 decoy ODN were transfected into U251 and A172 cells.2.Annexin V/PI dual staining assay was used to examine whether cell apoptosis was induced after STAT3 decoy ODN were transfected into U251 and A172 cells.3.RT-PCR assay was used to examine the mRNA expression of STAT3-targeted genes after STAT3 decoy ODN were transfected into U251 and A172 cells.4.Western blot analysis was used to examine the protein expression of STAT3-targeted genes after STAT3 decoy ODN were transfected into U251 and A172 cells.Results1.STAT3 decoy ODN resulted in cell cycle arrest from G1 to S phase.Twentyfour hours after treated with 50 nmol/L STAT3 decoy ODN,U251 and A172 cells both exhibited a significant increase,cells in G0/G1 phase from 49.58±1.09%to 54.89±1.35%in U251(P＜0.05),from 63.52±1.61%to 72.03±1.14%in A172 (P＜0.05),and accompanied by a distinguished decrease in S phase,from 42.36±1.50%to 37.89±0.67%in U251(P＜0.05),from 22.23±0.51%to 16.00±0.40%in A172(P＜0.05).The cells treated with the mutant control decoy ODN showed no obvious alterations.2.STAT3 decoy ODN induced apoptosis in glioma cells.Annexin V/PI staining assay showed that treatment with STAT3 decoy ODN could lead to a significant increase in apoptosis,from 5.44±0.50%to 23.57±9.76%in U251 cells(P＜0.05), and from 5.20±0.24%to 22.48±0.85%in A172 cells(P＜0.05).In contrast,mutant control decoy ODN only induced 6.81%and 6.16%apoptosis respectively(P＞0.05).3.STAT3 decoy ODN could down-regulate the expression of STAT3 targeted genes in glioma cells.As showed in RT-PCR,the mRNA levels of c-myc,cyclin D1, and bcl-xl in U251 cells were decreased by 52%,50%and 27.67%,respectively (P＜0.05).In A172 cells,the mRNA levels of c-myc,cyclinD1,and bcl-xl decreased by 46.67%,40.67%,and 28.87%respectively(P＜0.05).In contrast,the mutant control decoy ODN showed no effect on the mRNA levels of the malignant glioma cells(P＞0.05).As shown in Western bolt analysis,compared with control,the protein expression levels of cyclinD1 and bcl-xl in U251 cell decreased 47.33%and 85.66%, respectively(P＜0.05).The protein expression levels of cyclinD1 and bcl-xl in A172 cells were attenuated by 38.25%(P＜0.05)and 3.63%(P＞0.05),respectively.Conclusion1.STAT3 decoy ODN result in cell cycle arrest from G1 to S phase in glioma cells.2.STAT3 decoy ODN can induce apoptosis in glioma cells.3.STAT3 decoy ODN can down-regulate the expression of STAT3 targeted genes in human glioma cells.STAT3 decoy ODN may symphysially contribute to human glioma cells growth suppression via the marked downregulation of the downstream oncogenes,and subsequent cell cycle arrest from G1 to S phase and cell apoptosis.In conclution, targeted blocking STAT3 signaling pathway with a decoy oligonucleotide may potentially be used as an effective anti-glioma therapeutic approach.