The Role Of NF-κB Inhibitors On Glioma And Its Mechanism

BackgroundGlioma is the most common primary tumor of the central nervous system.It is characterized by high invasion,high recurrence and drug resistance.The comprehensive treatment mode of surgery combined with radiotherapy and chemotherapy is the main treatment for malignant glioma,and surgery is still the main treatment for low-grade glioma.Chemotherapy is currently used for the clinical treatment of gliomas,especially for the treatment of advanced gliomas and reduction of recurrence.However,chemotherapy is still not satisfactory for gliomas,especially for solid tumors.Therefore,searching for new anti-tumor drugs with high specificity,low toxicity and high efficiency is an important field in the research of glioma.NF-κB plays an important role in the development of tumor.It has been proved that the expression level of NF-κB in many kinds of solid malignant tumors,such as non small cell lung cancer,liver cancer,pancreatic cancer and glioma,is closely related to the inhibition of cancer cell apoptosis and the promotion of invasion and metastasis.The activation of NF-κB is in a continuous state in many tumor cells,including hematopoietic and solid tumors.The abnormal activation of NF-κB is considered to be one of the key mechanisms in a variety of malignant tumors.The activation of NF-κB pathway in glioma cells is not invariable,but is common to a variety of in vivo and in vitro factors.At present,further research on the development of NF-κB pathway inhibitors still needs to be studied.It is necessary to make further efforts to explore the role of NF-κB inhibitors in the development of glioma and the mechanism of action in the related treatment.On the basis of early high-throughput screening,the purpose of this study is to observe the effect of NF-κB inhibitor on the proliferation and apoptosis of human glioma cell lines and human glioma cells,and to reveal the role and mechanism of NF-κB inhibitor to inhibit the malignant biological behavior of glioma from the cell molecular biology level.Finally,we will use the animal model of orthotopic xenografts to test the anti glioma effect of NF-κB inhibitor,and to reveal the therapeutic effect of NF-κB inhibitor on glioma in vivo,so as to provide experimental basis for the clinical application of NF-κB inhibitor.Methods(1)U87,M059K,U251cells and primary glioma cells were treated with compound from the Target-selective Inhibitory Library(#L3500,Selleck Chemicals)for 24 hours.Subsequently,cell viability was detected by CCK-8.(2)NF-κB inhibitors,CAPE,JSH-23 and S4-A,were used as the research objects,and the human glioma cell lines,U87,M059K,U251 and human primary glioma cells were used asthe research models.(3)The effect of NF-κB inhibitor on the viability of glioma cells was detected by CCK-8,and the proliferation ability of glioma cells was detected by Brdu method.The apoptosis level of glioma cells was detected by Hoechst33342 staining reagent and TUNEL method.(4)Realtime PCR and Western blot were used to detect the mRNA and protein expression levels oftarget genes in glioma cells.(5)ELISA method was used to detect the effect of NF-κB inhibitor on the concentration of TNF-alpha factor,and the activity of Caspase-3/7 in glioma cells was detected by kit.(6)The effect of NF-κB inhibitor CAPE on the proliferation and apoptosis of human glioma cell U87,and the effect on tumor volume and histopathologic,also survivalwere investigated by the nude mouse glioma in situ xenograft model.Alsodetected the concentration of TNF-alpha factor,the activity of Caspase-3/7 and the phosphorylation level ofthe p65 NF-κB in nude mouse.ResultsTo identify drugs which can inhibit the proliferation of glioma cells,we screened a commercial kinase inhibitor library with 464 compounds from Selleck Chemicals.We found that several chemicals inhibit the proliferation of U87 cell lines significantly.T he top5 chemicals were CAPE(Cell Viability=0.161),Cryptotanshinone(Cell Viability=0.191),PF-5274857(Cell Viability=0.197),Pifithrin-μ(Cell Viability=0.201)and Tenovin-6(Cell Viability=0.202).CAPE was a NF-κB inhibitor and another two NF-κB inhibitors,JSH-23(Cell Viability=0.271)and S4-A(Cell Viability=0.252),although not in the top 5 chemicals,had a strong inhibitory effect on the proliferation of U87 cells.To verify this effect,we tested the inhibitory effect of the three NF-κB inhibitors on other two glioma cell lines(M059K and U251)and primary maligant glioma cells.All the three NF-κB inhibitors showed significant effects on the inhibition of glioma cells proliferation.After treatment with different NF-κB inhibitors for human glioma cells for 24h,CCK-8 test results showed that NF-κB inhibitors CAPE,JSH-23andS4-A could significantly inhibit the cell viability of human glioma cell lines U87,M059K and U251,and human primary glioma cells(p<0.05).CAPE inhibited the proliferation of U87 cells most significantly.The results of Brdu assay showed that NF-κB inhibitors,CAPE,JSH-23 and S4-A,could significantly inhibit the proliferation of human glioma cell lines U87,M059K,U251 and human primary glioma cells.The results showed significant differences(p<0.05),and the inhibition effect of CAPE on the proliferation of U87 cells was most significant.The results of Hoechst staining showed that the glioma cells in the blank control group were stained with blue fluorescence after Hoechst staining,and the morphology was round or oval,while the human glioma cell lines U87,M059K and U251,and human primary glioma cells were treated by different NF-κB inhibitors,CAPE,JSH-23 and S4-A respectively.Nuclear morphologic changed,including nuclear condensation,chromatin fragmentation,the staining clumps clustered and fluorescence enhanced also apoptotic bodies appeared,indicated that NF-κB inhibitors promoted the apoptosis of human glioma cells.The results of TUNEL detection showed that the percentage of apoptosis in human glioma cell lines,U87,M059K,U251 and human primary glioma cells was increased significantly after treatment with NF-κB inhibitors CAPE,JSH-23 and S4-A respectively,and the apoptotic proportion was highest in U87 cells after treatment with CAPE.(3)Following the use of different NF-κB inhibitors to treat human glioma cells for24h,the results showed that NF-κB inhibitor CAPE,JSH-23 and S4-A could significantly inhibit the synthesis of TNF-αmRNA in the human glioma cell lines U87,M059K and U251,and the release of TNF-αfactor.The results of t-test were consistent,with significant differences(p<0.05).NF-κB inhibitors,CAPE,JSH-23 and S4-A,could significantly promote the activation of Caspase-3/7 in human glioma cell lines,U87,M059K,U251 and human primary glioma cells,with significant differences(p<0.05).NF-κB inhibitors,CAPE,JSH-23 and S4-A could significantly inhibit the phosphorylation of the p65NF-κB in the human glioma cell linesU87,M059K and U251 as well as human glioma cells.All of them had significant differences(p<0.05).There were no effect on the phosphorylation level of STAT1.(4)After inoculation of human glioma U87 cells in the control group,the growth rate of the tumor was fast and the growth rate of nude mouse in the group of NF-κB inhibitor CAPE was relatively slow.From the beginning of the fourteenth day,the volume of glioma in the two groups of nude mouse was significantly different(p<0.05).With the prolongation of inoculation time,the volume difference of glioma in the two groups of nude mouse was gradually enlarged.After four weeks of inoculation,the volume of glioma in the control group was 251.3±41.4mm~3 and the volume of CAPE group was 91.3±9.36mm~3.The nude mouse in the control group died from the sixteenth day,and the nude mouse in the CAPE group died from the eighteenth day,after inoculation of glioma cells.At the twenty-eighth day,the survival rate of nude mouse in the control group decreased to 0%,the survival rate of the nude mouse in the CAPE group was 50%,and the difference was statistically significant(p<0.01).The results of histopathological examination showed that the glioma cells in the nude mouse of the control group were rich and dense,the morphology was normal,and the number of apoptotic cells was little,while the density of glioma cells in the nude mouse of the CAPE group was relatively reduced,the morphology of some cells was abnormal,the morphologic changes of nuclear pyknosis and other apoptosis appeared,and the partial tissue could be seen as necrosis.Following 28 days of inoculation of glioma cells,the concentration of TNF-αin the glioma tissue of NF-κB inhibitor CAPE group was 412.5±52.3pg/ml,significantly lower than that of the control group(p<0.05);The protein activity of Caspase-3/7 in the nude mouse of CAPE group increased to 451.2±61.9%,significantly higher than that of the control group(p<0.01);the phosphorylation level of the p65NF-κB of nude mouse in the CAPE group was significantly lower than that of the control group(p<0.05).Conclusion(1)NF-κB inhibitors,CAPE,JSH-23 and S4-A,identified by high-throughput screening,can significantly inhibit the proliferation of human glioma cells and promote the apoptosis of human glioma cells.First,the inhibitory effect of NF-κB inhibitors on the malignant biological behavior of human glioma cells is revealed,which provides an experimental basis for subsequent research.(2)NF-κB inhibitors can inhibit the activation of p65NF-κB in human glioma cells,thereby reduce the release of TNF-αand enhance the activation of Caspase-3/7,eventually inhibit the proliferation of human glioma cells and promote the level of apoptosis.(3)CAPE can significantly inhibit the tumor volume of glioma in nude mouse and induce apoptosis of glioma cells.The effect is related to inhibiting the release of TNF-α,promoting Caspase-3/7 activation and inhibiting the activation of NF-κB pathway.It shows that NF-κB inhibitor CAPE has the role of anti-malignant glioma in vivo,which is expected to provide evidence for the transformation of clinical application.