Anti-metastatic Effect Of ¹³¹I-BmK CT In Gliomas

Objective: Prepare for ¹³¹I labeled BmK CT and investigate its effect on U87 cell proliferation.Methods: BmK CT was labeled with ¹²⁵I/¹³¹I by indirect labeling method to form ¹²⁵I/¹³¹ I -BmK CT.After purification,the stability of ¹²⁵I/¹³¹ I -BmK CT was investigated.The glioma targeting of BmK CT was evaluated by fluorescence and iodine-131 labeled BmK CT.U87 cell proliferation was determined using the cell counting kit-8（CCK-8）and plat colony formation assay.Cell cycle and cell apoptosis were analyzed by flow cytometry.AO/EB double fluorescence staining method was used to detect the apoptotic cells.Western blot was used to evaluate the apoptotic proteins expression after drug treatment.Results: ¹²⁵I/¹³¹ I -BmK CT was successfully prepared and was stable both in PBS and FBS.BmK CT was a glioma targeting polypeptide with no influence on cell proliferation at concentrations of 0 to 1000μg/ml,however,a significant reduction in cell proliferation was observed when cells were treated with ¹³¹ I -BmK CT.¹³¹ I -BmK CT could increased the proportion of U87 cells in the S phase and the G2/M phase and decreased the proportion of cells in the G1 phase.¹³¹ I -BmK CT could also enhance the cell apoptosis.The apoptotic proteins expression was regulated by both BmK CT and ¹³¹ I -BmK CT.Conclusion: BmK CT was a glioma targeting peptide and had no effect on U87 cell proliferation in the definite concentration at 0 to 1000μg/ml.Not only could ¹³¹ I -BmK CT inhibit the proliferation of U87 cells,but also induced the cell apoptosis.Objectives: Buthus martensii Karsch chlorotoxin（BmK CT）can significantly inhibit glioma cell migration and invasion without affecting normal brain cells.Our previous studies demonstrated that BmK CT interacts with matrix metalloproteinase（MMP）-2 on the surface of glioma cells to inhibit tumor metastasis.However,the underlying molecular mechanism of this effect remains unclear;therefore,we aimed to determine the mechanism behind the effects of iodine 131(¹³¹ I )-labeled BmK CT on the invasion of U87 cells.Methods: The effects of the drug on U87 cell migration and invasion were investigated via transwell invasion experiments and wound-healing assays.The expression of MMP-2,MMP-9 and tissue inhibitor of metalloproteinase-2（TIMP-2）proteins was assessed by enzyme-linked immunosorbent assays（ELISAs）,Western blotting analysis and immunofluorescent assays.The enzyme activity of the MMP-2 and MMP-9 protein was evaluated via gelatin zymography.Results: The transwell and wound-healing assays indicated that BmK CT exposure significantly inhibited the invasion and migration of U87 cells in a dose-dependent manner（P<0.05）.Western blotting,ELISA,and immunofluorescent assays revealed that the expression of MMP-2 and MMP-9 proteins was down-regulated,while TIMP-2 protein expression was up-regulated after treatment with BmK CT（P<0.05）.Although the anti-metastasis effect of ¹³¹ I -BmK CT was better than BmK CT（P<0.05）,the expression of the proteins had no difference when compared with BmK CT（P>0.05）.The gelatin zymography results indicated that BmK CT exposure inhibited the enzyme activity of MMP-2/9,and ¹³¹ I -BmK CT was more effective（P<0.05）.Conclusions: The anti-metastatic effect of BmK CT in U87 cells was associated with the down-regulation of MMP-2 and MMP-9 expression and the up-regulation of TIMP-2 expression.Objective: ^99mTc labeled BmK CT was prepared and its potential as a tumor-specific agent for SPECT imaging of glioma was performed via a xenografted nude mouse model.Methods: BmK CT was linked with DTPA to form BmK CT-DTPA,followed by radiolabeling with ^99mTc.After purification from PD-10 column,the stability of ^99mTc-BmK CT in vitro was studied.SPECT imaging at different time points was performed both in normal rabbits and tumor bearing nude mice after intravenous injection of ^99mTc-BmK CT.At 4 h post-injection,one tumor bearing mouse was sacrificed to remove the tumor and major organs.Their relative radioactivity ratios were recorded by analyzing the regions of interest.Results: The radiochemical yield of ^99mTc-BmK CT was above 80% and its mean radiochemical purity was more than 99%.The radiochemical purity of purified ^99mTc-BmK CT was greater than 90% within 24 h both in PBS and FBS.SPECT images of ^99mTc-BmK CT in normal rabbits showed that the major radioactivity was accumulated in the liver,kidney and spleen,while other tissues including thyroid had very low level.^99mTc-BmK CT could be slowly cleared from the body through urinary system.The SPECT imaging in tumor bearing nude mice suggested that mildly increased SPECT signal intensity was found in the tumor site at 0.5 h post-injection and much higher was displayed at 1 h and 2 h,followed by the highest at 4 h.After that,the tumor SPECT signal descended steadily and could still be detected at 8 h post-injection.Conclusion: ^99mTc-BmK CT was successfully prepared with a high radiochemical purity and stability and could be used as a tumor-specific ligand for SPECT imaging of xenografted glioma model in vivo.