ABSTRACT
Aim To investigate the effect of m6A demethylase FTO inhibitor(FB23-2)on human glioblastoma stem cell activity. Methods The effects of FB23-2 and Temozolomide on GSC were detected by CCK-8 assay and neurosphere formation assay. The effect of FB23-2 on self-renewal of GSC was detected by limited dilution assay in vitro. The effect of FB23-2 on the proliferation of GSC was detected by EdU method. The effect of FB23-2 on apoptosis of glioblastoma stem cells was detected by flow cytometry. Results CCK-8 assay showed that FB23-2 could effectively inhibit the cell viability of GSC with IC50 values of 7.11 μmol·L-1 and 4.63 μmol·L-1,respectively. The size and number of GSC neural sphere in FB23-2 treatment group were significantly reduced compared with control group. In vitro limited dilution experiment showed that FB23-2 effectively inhibited the self-renewal ability of GSC. EdU incorporation experiment showed that compared with the control group,the treatment group decreased to(70.59±13.74)% and(50.33±4.53)%,respectively. The apoptotic rates of the treated group were(12.16±1.90)% and(16.77±1.17)% by flow cytometry. Conclusions FTO inhibitor FB23-2 can effectively inhibit GSC growth,self-renewal and the formation of neural sphere. In addition,FB23-2 can inhibit the proliferation of GSC and induce its apoptosis.
ABSTRACT
Hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcription factor that plays an important role in oxygen homeostasis. In response to low level of oxygen, subunit HIF-1alpha expression is upregulated and transactivates its target genes essential for energy metabolism, erythropoiesis and vascular development. HIF-1alpha is thought to be able to protect hypoxic cells from apoptosis or necrosis under ischemic and anoxic conditions, the major trauma factors that affect the recovery of brain and spinal cord injury. Here we report the construction of recombinant adenovirus vector overexpressing HIF-1alpha intended for gene therapy against desired neuronal injuries. The recombinant vector could be packaged and yielded significantly high viral titers at 2 x 10(13) CFU in HEK293T cells and good expression levels of HIF-1alpha when superinfected in Hela cells.
Subject(s)
Humans , Adenoviridae , Genetics , Blotting, Western , Cell Line , Genetic Therapy , Genetic Vectors , Genetics , HeLa Cells , Hypoxia-Inducible Factor 1, alpha Subunit , Genetics , MetabolismABSTRACT
OBJECTIVE@#To study the mechanisms of cultured neurons injury mediated by nitric oxide and free oxygen radical during hypoxia and oxidative stress.@*METHODS@#The cultured newborn rat neurons were treated with hypoxia, H2O2 and pretreated superoxide dismutase (SOD) respectively. We examined the content of NO, malonaldehyde (MDA), lactate dehydrogenase (LDH) and SOD in cultured supernatant.@*RESULTS@#Comparing with that of control group, the content of NO, LDH, MDA increased and the content of SOD decreased in hypoxia group and H2O2 group. The content between NO and SOD showed the negative correlation. Administration of 200 U/ml SOD before oxidative stress could efficiently decrease the release of NO, LDH and MDA in neurons. The content of NO, LDH and MDA manifested in positive correlation in each group.@*CONCLUSION@#Hypoxia and oxidative stress increased NO production which strengthen neurons injury induced by free radical. SOD played an important role in elimination of free oxygen radicals and protecting neurons from injury by NO.