Interleukin 24 Induces Growth Suppression And Chemotherapy Sensibility Of Malignant Glioma Cells In Vitro And In Vivo

Malignant gliomas are the most common brain tumors in humans, and current treatments fail to provide long-term management of these tumors. Gene therapy has developed swiftly along with the development of molecular biology and molecular genetics for the past few years. It established a new road for the therapy of malignant gliomas. Interleukin-24 is a new member of interleukin-10 family. Interleukin-24 is discovered overexpression in human melanoma during terminal differentiation. It is called melanoma differentiation-associated gene-7.Several groups have demonstrated that Ad-mda7 (using Ad-mda7) induces apoptosis in a wide range of cancer cells (lung, prostate, breast, ovary, pancreas), so, it has become candidate gene of gene therapy. In addition, there are some researches about combination of interleukin-24 and radiotherapy or chemotherapy. For example, Nishikawa first discovered interleukin-24 can enhance the radiosensitizing effect of non-small cell lung cancer (NSCLC) cell lines. Chada discovered the combination of interleukin-24 and radiotherapy or chemotherapy has synergistic action.Human glioma cell line U251 and glioma tissue were infected with a new type recombine adenovirus included interleukin-24 in this study. We have to observe the influence of interleukin-24 to glioma cell in vitro and in vivo. it was studied that Combination of interleukin-24 and chemotherapy medicine VM-26 is synergy or no synergy. We approached its potential mechanism of the action.Part one Construction of recombinant adenovirus Ad5F35-IL24Objective: to construct recombinant adenovirus Ad5F35-IL24.Methods: When plasmids of ATCC were cut by both HindIII and SalI enzymes, the goal bands were obtained. Plasmids of pDC316 were cut by both HindIII and SalI enzymes. Subsequently, after some fragments were connected, transformed and filtered, reorganized plasmids of pDC316 were obtained. Thereafter it was identified with PCR and enzymes cut. Then the plasmids were carried out sequence determination in whole automatic nucleic acid analyzer. Subsequently competent cells were prepared and transformed. After large-scale preparation of plasmid DNA, Recombined adenoviruses Ad5F35-IL24 were generated with homologous recombination by cotransfection of 293 cells with a couple plasmids. After it be identified with PCR, Ad5F35-IL24 recombinant adenovirus species was amplified and purified. Then we assayed its titre according to formula: VP/ml=OD₂₆₀×1.1×10¹² after it be identified with PCR.Results: Reconstructed Plasmids of pDC316-IL24 containing IL-24 gene was successfully constructed and identified by PCR, enzymes cut and sequence determination. Escherichia coli strains containing IL-24 gene was obtained. Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, amplified, purified and identified with PCR. Its titre was assayed according to formula: VP/ml=OD₂₆₀×1.1×10¹², and the titer was 2.8×10¹¹vp/ml.Conclusions: Reconstructed adenovirus Ad5F35-IL24 containing IL-24 gene with functional defect of proliferation was successfully constructed, and the titer was 2.8×10¹¹vp/ml.Part two Interleukin 24 induces growth suppression of malignant glioma cells in vitro and in vivoObjective: We have to observe the influence of interleukin-24 to glioma cell in vitro and in vivo. We approached its therapeutic effect to malignant glioma and potential mechanism of action.Methods: when human fetal astrocytes were primary cultured sucessfully, They and U251 cells were transfected with Ad5F35-IL24. influence of human fetal astrocytes and U251 cells was observed in vitro by Ad5F35-IL24.While animal models of human glioma were established and Ad5F35-IL24 was injected into tumor nodules in vivo, the tumor growth was observed. Their apoptosis and Bax/Bcl-2 expression were detected with flow cytometer and TUNEL. Results: IL-24 proteins were expressed highly after inflection of human fetal astrocytes, U251 cells and tumor tissues with Ad5F35-IL24. growth of human fetal astrocytes wasn't suppressed obviously(P>0.05). While growth of U251 cells and xenograft tumors was suppressed heavily, their apoptosis and expression of Bax/Bcl-2 were elevated significantly(P<0.05).Conclusion: Interleukin 24 selectively induces growth suppression and apoptosis in human glioma cells U251. Adenovirus-mediated IL24 gene therapy is a potential effective approach of malignant glioma.Part three Empirical study of mechanism about growth suppression of malignant glioma cells in vitro and in vivo by Interleukin 24Objective: to approach its potential mechanism of growth suppression of malignant glioma cells by Interleukin 24.Methods: After administration of 2-aminopurine to U251 cell, we detected inhibition rate with MTT, Apoptosis and expression of Bax/Bcl-2 protein with flow cytometer. We detected expression of IL-24, PKR, p-PKR, p38-MAPK, p-p38-MAPK, BAX, Bcl-2 protein of tumor tissues and U251 cell administrated and unadministrated by 2-aminopurine with western blot. Blood vessel density and expression of bFGF,VEGF would be assayed with immunohistochemistry,.Results: Western Blot analyses found that, IL-24 selectively induced U251 cell apoptosis by up-regulating and activating PKR protein(P<0.01), which would further activate p38MAPK pathway. After administration of 2-aminopurine blocked action of PKR protein, apoptosis and action of p38-MAPK were blocked. These further proved mechanism of action. We found that vascularization and expression of VEGF, bFGF is reduced obviously in tumor administrated IL-24 with Immunohistochemistry(P<0.05).Conclusions: IL-24 selectively induced U251 cell and tumor apoptosis by up-regulating and activating PKR protein, which would further activate p38MAPK pathway. IL-24 inhibits glioma angiogenesis, thus inhibits tumor proliferation. The mechanism may involve the reduction of expression of VEGF and bFGF by IL-24 in gliomas. Part four Empirical study on effect of chemotherapy sensibility in malignant glioma cells in vitro and in vivo by Interleukin 24Objective: To observe that Combination of interleukin-24 and chemotherapy medicine VM-26 is synergy or no synergy and approach its potential mechanism of the action.Methods: After administration of IL-24 and VM-26 to U251 cell, we detected inhibition rate with MTT,Apoptosis with Hochest33258 and expression of Bax/Bcl-2 protein with flow cytometer. We detected expression of IL-24, PKR, p-PKR, p38-MAPK, p-p38-MAPK, BAX, Bcl-2 protein of U251 cell administrated by IL-24 and VM-26 with western blot. Apoptosis was assayed in all groups of tumors with TUNEL. expression of BAX, Bcl-2 protein was detected in all groups of tumors with flow cytometer. Blood vessel density and expression of bFGF,VEGF would be assayed with immunohistochemistry.Results: After administration of IL-24 and VM-26 to U251 cells and tumor tissues, inhibition rate of U251 cells was significantly raised up with MTT(P<0.001), Apoptosis of U251cells and tumor tissues was significantly raised up with Hochest33258 and TUNEL(P<0.001), expression of Bax/Bcl-2 protein was significantly raised up with flow cytometer(P<0.05), expression and action of PKR and p38MAPK in U251 cells were significantly raised up with western blot(P<0.001). vascularization and expression of VEGF, bFGFo is reduced obviously in tumor administrated by IL-24 and VM-26 with Immunohistochemistry(P<0.05).Conclusions: L-24 and VM-26 synergisticly inhibit the proliferation and induce the apoptosis of U251 cells and glioma, which may be related to the apoptosis pathway and the increased ratio of the pro-apoptotic protein Bax and Anti-apoptosis protein Bcl-2. IL-24 and VM-26 inhibit glioma angiogenesis, thus inhibit tumor proliferation. The mechanism may involve the reduction of expression of VEGF and bFGF in gliomas. The experiment showed that combination administration of IL-24 and VM-26 are all desirable treatments for gliomas.