Experiment Investigation On Radiosensitizing Effect Of CpG ODN107and Its Mechanisms On Human Glioma U87Cell Line

Objective:Glioma is the most common subtype of primary brain tumors with high morbidity,high recurrence rate, high mortality and low recovery rate. Surgery in combination withsubsequent radiation therapy is the standard treatment for glioma due to glioma is invasiveand cannot be completely excised. However, glioma cells become insensitive to radiation orreduced sensitivity to radiation after a period of therapy, radiosensitizer can improve thesensitivity of tumor cells to radiation and enhance the effect of radiotherapy. Therefore, it issignificant to search for a radiosensitizer to improve radiotherapy.NO was thought to be the best effective radiosensitizer. Under the condition ofhypoxia, NO can improve the sensitivity of tumor cells to radiation, but it has obviousneurotoxicity for human. The main mechanisms of radiosensitizer are related to cell cyclearrest, apoptosis, autophagic death, etc.CpG ODN, containing unmethylated CpG motifs oligodeoxynucleotides, is thesmallest unit of immunostimulatory effects of bacterial DNA. Recent years, CpG ODN as anew radiosensitizer was extensively investigated. The experimental results confirmed thatradiosensitizing effect of CpG ODN is related to activation of NK cells, release of NO andTNF-α. But more mechanism should be investigated.Our present work is one important part of major scientific and technological specialproject for “Significant New Drugs Creation”of china (2009ZX09103-051). CpG ODN107is a newly identified oligodeoxynucleotides in our lab; we investigated radiosensitizingeffect and mechanism of CpG ODN107for human glioma U87Cell line. This experimentprovides the experimental basis of looking for the efficient, low toxicity radiosensitizerdrugs, and new ideas for glioma treatment.Research Contents and Methods: I. Experiment investigation on radiosensitizing effect of CpG ODN1071. Radiosensitizing effect of CpG ODN107in vitro(1) The viability of U87cells treated with different dose of β-ray irradiation (0,2,4,6,8,10Gy) or five gradient concentrations (ranging from1to90μg/ml) of CpG ODN107orCpG ODN107in combination with β-ray irradiation was detected using MTT assayexperiment.(2) Colony formation experiment was carried out to evaluate the radiosensitizing effectof CpG ODN107in combination with irradiation in vitro.2. Radiosensitizing effect of CpG ODN107in vivoIn order to confirm whether CpG ODN107possessed the potential radiosensitizingeffect for glioma cells in vivo, CpG ODN107was evaluated in nude mice glioma xenograftmodel and orthotopic model.II. The effect of CpG ODN107in combination with irradiation on the U87cellproliferation and death1. Cell cycle was detected with flow cytometry (FCM)2. Cell apoptosis was detected with flow cytometry (FCM)3. The effect of CpG ODN107in combination with irradiation on U87cell aotuphagy(1) Transmission electron microscopy was used to observe the autophagosomeformation in U87cells treated with CpG ODN107in combination with irradiation.(2) The effect of CpG ODN107in combination with irradiation on U87cells GFP-LC3aggregation.(3) The effect of CpG ODN107in combination with irradiation on autophagy markerprotein LC3and beclin-1expression by Western blot.III. The radiosensitization CpG ODN107of the preliminary study of the mechanism ofaction. The effect of CPG ODN107in combination with irradiation on U87gliomaradiosensitizing effect signal pathway1. The effect of CpG ODN107on TLR9/NF-κB signal pathway(1) NO level was tested by Griess method(2) iNOS expression was tested by ELISA method(3) TLR9mRNA expression was testd by Rea-time PCR(4) NF-κB activation was detected by ELISA method 2. The effect of CpG ODN107on HIF-1α/VEGF signal pathway(1) CD34expression in brain tumor tissues was observed by immunohistochemistry,and then MVD of tissue sections was evaluated according to Gasparini’s criteria.(2) VEGF expression in U87cells and in brain tumor tissues was tested by ELISAmethod(3) HIF-1α expression in U87cells was tested by Western blot and HIF-1α expressionin brain tumor tissues was observed by immunohistochemistry, respectively.Results:I. Investigation on radiosensitizing effect of CpG ODN1071.10μg/mL of CpG ODN107significantly improved the sensitivity of U87cells toβ-ray.2.10μg/mL of CpG ODN107in combination with irradiation significantly inhibitedcell proliferation both in MTT assay and colony formation experiments.3. The inhibition ratio of tumor growth produced by CpG ODN107(1.7,5, and15mg/kg) in combination with irradiation was27.3,67.0, and65.5%, respectively.CpGODN107in combination with β-ray irradiation apparently decreased the growth of the U87glioma xenografts in nude mice.4. Three concentrations of CpG ODN107(0.75,0.25and0.083mg/kg) in combinationwith irradiation prolonged the median survival of orthotopic model (23days,28days, and37.5days, respectively). CpG ODN107(0.083mg/kg) in combination with irradiationincreased nude mice survival rate.II. The effect of CpG ODN107in combination with irradiation on the cell cycle,apoptosis, autophagy death of human glioma U87Cell line CpG ODN1071. CpG ODN107in combination with irradiation induced cell cycle arrest at G1phase.2CpG ODN107in combination with irradiation did not induce apoptosis.3. CpG ODN107in combination with irradiation induced U87cell aotuphagy death.(1) CpG ODN107in combination with irradiation increased the autophagosomeformation.(2) CpG ODN107in combination with irradiation induced GFP-LC3aggregation. (3) CpG ODN107in combination with irradiation increased autophagy marker protein.LC3and beclin-1expression.III. Investigation of radiosensitizing mechanisms1. The effect of CpG ODN107on TLR9/NF-κB signal pathway(1) CpG ODN107in combination with irradiation increased NO level of U87cell.(2) CpG ODN107in combination with irradiation increased iNOS expression.(3) CpG ODN107in combination with irradiation upregulated TLR9mRNAexpression.(4) CpG ODN107in combination with irradiation increased NF-κB activation.2. The effect of CpG ODN107on HIF-1α/VEGF signal pathway(1) CpG ODN107in combination with local radiotherapy upregulated CD34expression and decreased MVD in vivo.(2) CpG ODN107in combination with irradiation decreased VEGF expression in vitroand in vivo.(3) CpG ODN107in combination with irradiation upregulated HIF-1α expression invitro and in vivo.Conclusions:1. CpG ODN107potentiates radiosensitivity of human glioma U87cell lineCpG ODN107(10μg/ml) in combination with irradiation significantly inhibited cellproliferation in MTT assay and colony formation experiments.CpG ODN107(5mg/kg) in combination with irradiation apparently decreased thegrowth of the U87glioma xenografts in nude mice.CpG ODN107(0.083mg/kg) in combination with irradiation prolonged nude micesurvival in orthotopic model.2. CpG ODN107in combination with irradiation did not induce apoptosis but inducedcell cycle arrest at G1phase and autophagy cell death in U87cell. The results demonstratedthat CpG ODN107possessed a radiosensitizing effect related to cell cycle arrest at G1phaseand autophagic cell death.3. CpG ODN107played its radiosensitizing effect via activated TLR9/NF-κB pathway leading to NO production in tumor cells an via HIF-1α/VEGF pathway leading to inhibitionof tumor angiogenesis. These two pathway coenhanced the radiosensitivity of tumor cellleading to tumor suppression and prolonging survival period.