Dimethyl Phenyl Piperazine Iodide (DMPP) Induces Glioma Regression By Inhibiting Angiogenesis

ObjectiveTo investigate the inhibitory effects of human malignant glioma U87cells onchicken embryo membrane(CAM) growth after treatment with1,1-Dimethyl-4-phenyl piperazine iodide (DMPP), as well as DMPP’s inhibitory effects onangiogenesis and the angiogenic-associated signaling genes, and illustrate its possiblemechanism.MethodChicken embryo membrane (CAM) xenograft model: CAM widely used toexplore the role of tumor tissue played related with angiogenesis. Using thisxenograft model, we investigated whether DMPP could inhibit the growth of U87cells, together with effects on angiogenesis.Hematoxylin and eosin staining (H&E staining): To observe organizationalstructure and cell morphology dyeing.Cell culture in vitro: Cultivate the human malignant glioma cell line U87cells invitro for xenograft.5-Bromo-2-deoxyUridine (BrdU) assay: chick CAM and U87cells wereincubated with5-bromo-20-deoxyuridine (BrdU; Sigma-Aldrich) to investigate theextent of cell proliferation in these cells/tissues.(3-4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)MTT: Todetermine the cell activity and cell proliferation.Western blotting: To check whether DMPP exposure will affect the apoptosis ofU87cells, western blotting was performed on the expression of PARP, Caspase-9,Caspase-3and Bcl-2in presence of various concentrations of DMPP.Flow cytometry (propidium iodide,PI): Detection of U87cell apoptosis afterDMPP treatment.Masson’s trichrome staining: To reveal the presence of fibrosis in sections ofchick and cancer tissues.Immunofluorescence: combined with FITC SNA-1lectin to mark vascularendothelial cells.Whole-mount in situ hybridization: VE-cadherin probe was synthesized via molecular biology methods. Endogenous expression of VE-cadherin was firstlyidentified through in situ hybridization.Chicken embryo yolk sac membrane(YSM) model: YSM assay was used todetermine the effect of DMPP on angiogenesis.RT-PCR (Reverse transcription-PCR): After DMPP treatment all the RNA wasextracted from CAM tissues, and the effects of DMPP on angiogenic-associatedsignaling genes were examined by RT-PCR, according to the manufacturer’sinstructions.ResultIn this study, we demonstrated that DMPP could dramatically inhibit glioma sizemaintained on chick embryonic chorioallantoic membrane (CAM). We firstperformed MTT、BrdU and Flow cytometry incorporation experiments on U87glioma cells in vitro to understand the mechanism involved. Then, we found out thatDMPP had no significant effect on the proliferation and survival of U87cell. Hence,we performed detail analysis of DMPP’s inhibitory effects on angiogenesis. Threevasculogenesis and angiogenesis in vivo models were used in the study whichincluding early chick blood islands formation, chick yolk-sac membrane and CAMmodels. The results revealed that DMPP can directly suppress all developmentalstages involved in vasculogenesis and angiogenesis. We investigated the effect ofDMPP on angiogenic-associated signaling genes in chick CAM by RT-PCR, wefound DMPP treatment significantly repressed Ang-1and HIF-2α expression.ConclusionIn sum, our results show that DMPP could induce glioma regression incubatedon CAM by inhibiting vasculogenesis and angiogenesis. In addition, the impairmentof Ang-1and HIF-2α signaling may play a pivotal role on how DMPP’s inhibitory onangiogenesis.