Inhibitory effect of gamma-secretase inhibitor on retinal neovascularization and regulative mechanism of Notch1 signal pathway / 中华实验眼科杂志

Objective:To investigate the inhibitory effect and underlying mechanism of gamma-secretase inhibitor blocking Notch1 signaling on retinal neovascularization caused by oxygen-induced retinopathy (OIR) in mice.Methods:To establish the OIR model, 7-day-old pups of C57BL/6J mice were exposed to 75% oxygen together with their mother until postnatal day (P)12.On P12, the mice were transferred to room air.All the mice were randomly divided into three groups, OIR group as control group, OIR+ DAPT group and OIR+ DMSO group receiving 1 μl intravitreal injection of gamma-secretase inhibitor (DAPT, 10 mmol/L) and 1∶20 DMSO dilution respectively.The right eye was taken as experimental eye.The mice were euthanized on P17 and the eyes were harvested to obtain retinas for further investigation.The total proteins were extracted from the retinas.The relative expression levels of Notch1 signal pathway and its downstream Hes1, the markers of M1 phenotype inducible nitric oxide synthase (iNOS) and M2 phenotype arginase-1 (Arg-1) microglia were measured by western blot.Retinal flat mounts were made and the retinal vessels were stained with isolectin B4 (IB4) to investigate the relative retinal neovascularization areas which was calculated as the ratio of neovascularization area/retinal area.The mumber of the neovascular endothelium cells beyond the inner limiting membrane was observed by hematoxylin-eosin staining.The use and care of animals complied with ARVO statement.This study protocol was approved by the Animal Ethics Committee of Guangdong Provincial People's Hospital (No.KY-Z-2021-2015-01).Results:The relative protein expression levels of Notch1 and Hes1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.68±0.06 and 0.70±0.08, 1.00±0.00 and 1.00±0.00, 1.03±0.08 and 1.02±0.07, respectively, with statistically significant differences among them ( F=70.62, 53.65; both at P<0.01). Compared with the OIR group and OIR+ DMSO group, the expressions of Notch1 and Hes1 were significantly reduced in OIR+ DAPT group (all at P<0.01). The relative protein expression levels of iNOS and Arg-1 in OIR+ DAPT group, OIR group, and OIR+ DMSO group were 0.74±0.07 and 1.49±0.12, 1.00±0.00 and 1.00±0.00, 1.04±0.10 and 0.94±0.07, respectively, showing statistically significant differences ( F=31.63, 89.32; both at P<0.01). Compared with OIR group and OIR+ DMSO group, the expression of iNOS in OIR+ DAPT group was significantly reduced, and the expression of Arg-1 was significantly increased (all at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells beyond the inner limiting membrane in OIR+ DAPT group, OIR group, and OIR+ DMSO group were (8.82±2.71)% and 38.17±3.29, (22.32±5.34)% and 60.83±5.11, (20.27±3.36)% and 58.67±4.75, respectively, showing statistically significant differences ( F=33.72, 39.44; both at P<0.01). The relative neovascularization area and the number of neovascular endothelium cells in OIR+ DAPT group were significantly reduced in comparison with OIR group and OIR+ DMSO group (all at P<0.01). Conclusions:Intravitreal injection of DAPT can inhibit the retinal neovascularization in OIR mice through blocking Notch1 signaling activation and promoting retinal microglia polarization from M1 to M2 phenotype.

Alteration of NOTCH1 in T-cell Acute Lymphoblastic Leukemia and Development of Target Therapeutic Agent / 임상소아혈액종양

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of entire ALL in children. The outcome of T-ALL has been improved through the intensified therapeutic strategy, however, it is still a more aggressive disease. In T-ALL a couple of transcription factor oncogenes are known to be relocated to the juxtaposition of T-cell receptor genes, potent promoter, by chromosome translocation. However the incidence of each chimeric gene formation in T-ALL is less than 5% and their clinical significance as a prognostic marker is lacking. A decade ago it was identified that activating mutations in NOTCH1 in about 60% of T-ALL. After then, activating NOTCH1 mutations present in T-ALL have been extensively investigated with regard to understanding its molecular pathogenesis, its prognostic significance, and developing molecularly tailored novel agents. Small molecule gamma-secretase inhibitor, blocking a proteolytic step required for creation of a fragment of NOTCH intracellular domain which actually act as a controller of its target gene expression, was tried as a target therapeutic drug for T-ALL. Although outcome of this drug was not satisfactory, challenges have been launched to develop new drugs which specifically act on the aberrant behavior of mutated NOTCH1 in T-ALL.

Alteration of NOTCH1 in T-cell Acute Lymphoblastic Leukemia and Development of Target Therapeutic Agent / 임상소아혈액종양

T-cell acute lymphoblastic leukemia (T-ALL) accounts for approximately 10-15% of entire ALL in children. The outcome of T-ALL has been improved through the intensified therapeutic strategy, however, it is still a more aggressive disease. In T-ALL a couple of transcription factor oncogenes are known to be relocated to the juxtaposition of T-cell receptor genes, potent promoter, by chromosome translocation. However the incidence of each chimeric gene formation in T-ALL is less than 5% and their clinical significance as a prognostic marker is lacking. A decade ago it was identified that activating mutations in NOTCH1 in about 60% of T-ALL. After then, activating NOTCH1 mutations present in T-ALL have been extensively investigated with regard to understanding its molecular pathogenesis, its prognostic significance, and developing molecularly tailored novel agents. Small molecule gamma-secretase inhibitor, blocking a proteolytic step required for creation of a fragment of NOTCH intracellular domain which actually act as a controller of its target gene expression, was tried as a target therapeutic drug for T-ALL. Although outcome of this drug was not satisfactory, challenges have been launched to develop new drugs which specifically act on the aberrant behavior of mutated NOTCH1 in T-ALL.

Gamma secretase inhibitor-I exerted cytotoxic effects on malignant glionm cell lines by inducing cell cycle arrest and apoptosis / 中华神经医学杂志

Objective To investigate the role of gamma secretase inhibitor-I (GSI-I) in cell proliferation and apoptosis of human glioma cell lines U87 and U251.Methods RT-PCR and fluorescent quantitative RT-PCR (qRT-PCR) were employed to evaluate the expressions of Notch receptors and their target gene Hes-I in both U87 and U251 cells treated by GSI-I,respectively.Then,MTT assay was used to examine the effects of GSI-I on cell proliferation of the 2 glioma cells.Meanwhile,flow cytometry technique was also employed to detect the cell cycle changes and apoptosis induced by GSI-I treatment.Results The activity of Notch pathway was inhibited by GSI-I treatment through down-regulating the expression of Notch receptors target gene Hes-I in both U87 and U251 cells.Treatment with 2.5μmol/L GSI-I or above concentrations could significantly induce the cell cycle arrest of U87 and U251 cells and these effects were positively concentration-dependent.Flow cytometry technique showed that GSI-I inhibited the cell proliferation by inducing the cell cycle arrest of U87 cells at GI phase and inducing the apoptosis of U251 cells.Conclusion GSI-I can dramatically inhibit the cell proliferation and induce the apoptosis of U87 and U251 cells,providing a reliable evidence for clinical glioma treatment.