ABSTRACT
Malignant glioma is usually accompanied by vigorous angiogenesis to provide essential nutrients. An effective glioma targeting moiety should include excellent tumor-cell homing ability as well as good neovasculature-targeting efficiency, and should be highly resistant to enzyme degradation in the bloodstream. The phage display-selected heptapeptide, the glioma-initiating cell peptide (GICP), was previously reported as a ligand for the VAV3 protein (a Rho-GTPase guanine nucleotide exchange factor), which is mainly expressed on glioma cells; the stabilized heptapeptide A7R has been shown to be the ligand of both vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1), and has demonstrated good neovasculature-targeting ability. By linking A7R and GICP, a multi-receptor targeting molecule was obtained. The stability of these three peptides was evaluated and their targeting efficiency on tumor-related cells and models was compared. The ability of these peptides to cross the blood--tumor barrier (BTB) was also determined. The results indicate that the coupled Y-shaped peptide A7R-GICP exhibited improved tumor and neovasculature targeting ability and had higher efficiency in crossing the BTB than either individual peptide.
ABSTRACT
Objective To study the mechanism of miR?200c in regulating RMP7?induced increases of blood?tumor barrier(BTB)permeability by targeting Ras homolog gene family member A(RhoA). Methods Endogenous expression of miR?200c was detected by real?time PCR in hu?man cerebral microvascular endothelial cell line hCMEC/D3(ECs)after RMP7 treatment. miR?200c mimic and miR?200c inhibitor were transfect?ed into GECs(ECs with U87 glioma cells co?culturing),respectively. Transfection efficiency of miR?200c mimic and miR?200c inhibitor were de?termined by real?time PCR. HRP flux and TEER assays revealed BTB permeability. The protein expression level of RhoA was assessed by West?ern blotting. The distribution of RhoA was assessed by immunofluorescence microscopy. RhoA luciferase assays were performed using the Dual?Lucif?erase reporter assay system. Results RMP7 significantly induced a decrease in miR?200c expression in GECs of BTB. miR?200c mimic and miR?200c inhibitor were successfully transfected into GECs. Overexpression of miR?200c inhibited endothelial leakage and restored normal transendo?thelial electric resistance values. Simultaneously ,overexpression of miR?200c significantly reduced the protein expression level of RhoA. In addi?tion,immunofluorescence analysis revealed that the distribution of RhoA in the cytoplasm and nuclei of GECs were decreased in miR?200c mimic group. RhoA was one of the direct targets of miR?200c with the specific binding site being located at the seed sequence. The results of miR?200c si?lencing were opposite to that of the miR?200c overexpression group. Conclusion miRNA?200c regulated RMP7?induced increases in BTB perme?ability by targeting RhoA.
ABSTRACT
Aim To study the effect of IL-1β on pro-tein expression of vascular endothelial growth factor in glioma cells and plasma membrane microcapsule struc-ture protein caveolin-1 and plasma membrane vesicles in brain microvascular endothelial cells, and prelimi-narity discuss the possible mechanism of IL-1β opening blood tumor barrier. Methods The tumor barrier mod-el was established by transwell in vitro. The effect of IL-1β on the expression of VEGF in glioma cells and caveolin-1 in brain microvascular endothelial cells was dynamically monitored by Western blot. TEM was used to observe the number of plasma membrane vesicles of brain microvascular endothelial cells. Sodium fluores-cein leakage test was used to assess the permeability of blood tumor barrier after IL-1β. Results The tumor barrier model was successfully established by transwell in vitro. When IL-1β treated the model of blood tumor barrier,the expression of VEGF increased,and reached the peak at 60min,and recovered to the initial state at 120min. The permeability of the blood tumor barrier model was the highest at 60min. In addition,our re-sults also found that,the protein expression of plasma membrane microcapsule structure protein caveolin-1 and number of plasma membrane vesicles in brain mi-crocapsule endothelial cells reached peak at 60 min, subsequently reduced and returned to non drug state at 120min. Conclusion IL-1β increases blood tumor barrier permeability,which may be related to IL-1β in-creasing the number of plasma membrane vesicles through VEGF/ caveolin-1 pathway.
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Aim To investigate the signaling mecha-nisms in endothelial monocyte-activating polypeptide-Ⅱ( EMAP-Ⅱ)-induced increase in blood-tumor barri-er ( BTB ) permeability. Methods Relatively pure cerebral microvessel fragments were obtained from the cortex of 3-5 days old Wistar rats by using careful dis-section, enzyme digestion, and dextran centrifugation. Then, these fragments were seeded on dishes and cul-tured primarily. In vitro BTB models were constructed by co-cultivation of rat brain microvascular endothelial cells ( BMECs) with C6 glioma cells. Confluent mono-layers of co-cultured BMECs were divided randomly in-to 5 groups ( each n=6 ): control, EMAP-Ⅱ, H7 +EMAP-Ⅱ, C3 exoenzyme + EMAP-Ⅱ, and C3 ex-oenzyme + H7 + EMAP-Ⅱ groups. Transendothelial electric resistance values and horseradish peroxidase flux were measured to evaluate changes in the BTB permeability . The expression levels of tight junction-re-lated protein occludin and ZO-1 in BMECs were meas-ured by Western blot. Immunofluorescence was used to identify the expression and distribution of occludin and ZO-1 in BMECs. Also, Western blot were used to de-tect the expression levels of myosin light chain ( MLC) and phosphomyosin light chain ( pMLC ) in BMECs. Results Compared with control group, the BTB per-meability of EMAP-Ⅱ group was increased significant-ly. The expression levels of occludin and ZO-1 in BMECs were significantly decreased, accompanied with marked increase in the expression level of pMLC. These above-mentioned effects of EMAP-Ⅱ were sig-nificantly inhibited by pretreatment with H7 ( an inhib-itor of PKC ) or/and C3 exoenzyme ( an inhibitor of RhoA ) . Conclusion Signaling molecules PKC and RhoA play important roles in EMAP-Ⅱ-induced in-crease in BTB permeability; signaling pathways PKC-pMLC and RhoA-pMLC are involved in this process.
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Aim To investigate whether the annexation of bradykinin and papaverine had some synergetic effects on the opening of blood-tumor barrier,and find out the action of cytokine in this mechanism.Methods 160 female rats were divided into 8 groups:sham operated group;model group,PA group,BK group,PA+BK group,1/2PA+BK group,1/2BK+PA group and 1/2PA+1/2BK group.20 rats were needed in each group.The C6 brain tumor model was built.The drug was pumped into rat's brain via the carotid artery.The opening of the tight junction was detected by electron microscop.And the permeability of the blood-tumor barrier was tested by Evans blue.The expression of IL-1? was tested by Western blot and the expression of IL-1? on/around the capillary in the brain of the cerebral glioma rats was tested by SABC immunochemistry.Results In contrast to sham operated group/model group,the expression of IL-1? in PA group,BK group and PA+BK group increased obviously(P
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Aim To assess the effect of heat shock factor-1(HSF1)and heat shock protein 70(HSP70)to tachyphylaxis by bradykinin in the opening blood-tumor barrier(BTB).Methods The C6 rat intracerebral glioma model was constructed by stereotactic implantation technique.Using western blotting method to continue monitoring the protein expression of HSF1 and HSP70 in tumor tissue after bradykinin acted on animals.The expression of occludin in tumor tissue were detected by immunohistochemistry method.Using Evans blue and electron microscope,respectively,detected the permeability and pathology of BTB after intracarotid infusion of bradykinin in C6 rats.Results Bradykinin increased the tight junction opening and the permeability of BTB in C6 animals,and the relative increments of EB were 5.19 ?g?g-1(0 min),5.06 ?g?g-1(5 min),11.35 ?g?g-1(10 min,P
ABSTRACT
Aim To investigate the effects of bradykinin on the expression of IL-1? in C6 glioma cells and provide an experimental basis for the mechanism of bradykinin opening the blood tumor barrier.Methods Semi-quantitive RT-PCR and radioimmunoassay were used to detect the expression of IL-1?mRNA in C6 glioma cells and the content of IL-1? in the culture media treated with bradykinin respectively.Results RT-PCR revealed that the expression of IL-1?mRNA was significantly elevated with bradykinin treated for 15min(P
ABSTRACT
Objective To study the morphological characteristics of blood-tumor barrier(BTB)model of glioma in vitro. Methods After C 6 glioma cells/endothelia ECV 304 co-cultured mixed or in Transwell or on both sides of membrane of Transwell,the morphological characters of fenestra of endothelial cells,the junction between ECV 304 cells,the interface between C 6 cells and ECV 304 cells,and perivascular-end-feet of C 6 cells were observed under transmission electron microscope(TEM)and scanning electron microscope(SEM),and compared to BTB in 4 cases with human brain glioma. Results ECV 304 cells grown to confluence were not fenestrated,but with fomation of tight junction between ECV 304 cells after co-cultured with C 6 cells mixedly,in Transwell or on both sides of membrane of Transwell;It was not found that pseudopodia from C 6 cells as co-cultured in Transwell reaching into pore of Transwell;C 6 cells co-cultured on both sides of membrane of Transwell often sticked out to the ECV 304 cells,but with no pseudopodia from C 6 cells surrounded ECV 304 cells of penetrated the endothelia clefts.Perivascular-end-feet of C 6 cells were not integrant.These characters were similar to BTB in human brain glioma.Conclusion C 6 glioma cells/endothelia cell ECV 304 co-cultures on both sides of membrane of Transwell may simulate the morphological characters of BTB in vivo in some degree.;