The Reg?lation And Mechanism Of The Dual PI3K/mTOR Signaling Inhibitor In Hemangioma Vasc?lar Endothelial Cell In Vitro

Objective To observe the influence of the dual PI3K/m TOR inhibitor NVP-BEZ235 on the cell proliferation and apoptosis of hemangioma in vitro and key molec?les of the PI3K/AKt/m TOR signaling pathway, to investigate the reg?lation and mechanism of the dual PI3K/m TOR inhibitor in hemangioma vasc?lar endothelial cell in vitro.Methods 1. The hemangioma-derived endothelial cells(Hen ECs) were obtained by surginal resection, and c?ltured by explant combined with trypsin-digestion mothod. Cells were draw the growth curve by the observation of the growth characteristic and identified the types of cells by Aactor VIII antigen, which co?ld exist in Hen ECs specifically. 2.The fourth generation cells were c?ltured with ser?M free medi?M for 24 hours,of which the intervention group were added to the 0.50 ?M,1.00 ?M NVP- BEZ235, the blank control group were added to the c?lture medi?M. After being incubated for 24 h, the cells were detected the proliferation with CCK-8, the cell apoptosis with flow cytometry, the protein expression of PI3 K,AKt,m TOR and p70s6 k with Western blot, and analyzed the relationship between the proliferation and apoptosis of hemangioma vasc?lar endothelial cells and the expression level of the four proteins.Res?lts 1. The Hem ECs migrated from the hemangioma tissue after the 1st week, the growth rate spred and shapd the concentric circle distribution in the next three weeks, then cells basically covered the bottom of c?lture bottle in the 4th week, we observed the spindle shaped or slightly irreg?lar cells. And the cells of Factor VIIIantigen were apparently positive. 2. The Hem ECs were intervened with 0.50 ?M, 1.00 ?M NVP-BEZ235 for 24 hours. The CCK-8 showed that the cell proliferation OD of 0.50 ?M, 1.00 ?M NVP-BEZ235 group were(0.88±0.03,0.59±0.05),comparised with the OD of control group was(1.10±0.02),thedifferences were statistically significant in the inhibition of hemangioma vasc?lar endothelial cell(P<0.01, t=7.05,13.07); furthermore,the inhibitory effect on cell proliferation of1.00 ?M NVP-BEZ235 group was stronger than that of 0.50 ?M NVP-BEZ235 group, and there was significant difference(P<0.01, t=9.23). 3. The cells were treated with 0.50 ?M, 1.00 ?M NVP-BEZ235 for 24 hours, the rate of G0/G1 phase cells are 0.50 ?M NVP-BEZ235RAD001(60.62 ± 0.71)%, 1.00 ?M NVP-BEZ235(65.99±2.55)%, which was higher than the rate of the control group(53.71±1.43)%, and the differences were statistically significant(P<0.01, t=-7.51,-7.28)). 1.00 ?M NVP-BEZ235 group was stronger than that of 0.50 ?M NVP-BEZ235 group(P<0.05, t=-3.51). 4.Flow cytometry showed the total rate of apoptosis cells of 0.50?M,1.00?MNVPBEZ235group were(9.2±0.75)%,(13.1±1.72)%, two groups were higher than the blank control group(2.77±1.23)%, and the differences were significant(P<0.01, t=-9.80,-8.93); moreover,the 1.00?MNVP- BEZ235 group was higher than the 0.50?M NVPBEZ235 group, the differences were statistically significant(P<0.05, t=-3.62). 5. By western blot, the protein level of PI3K(0.16±0.03), p-Akt(0.13±0.01)and m TOR(0.12±0.02)in the 0.50?M NVP- BEZ235 group and the protein levels of PI3K(0.10±0.01),p-Akt(0.10±0.01),m TOR(0.05±0.00) in the 1.00?M NVP- BEZ235 group, were both lower than the protein levels of PI3K(0.25±0.01), p-Akt(0.17±0.01)and m TOR(0.19±0.00) in the blank control group, the differences were both significant(P<0.01, t=4.71,8.51,6.62,26.68,15.40,38.84); the protein level of P70S6K(0.18±0.01) in the 0.50?M NVP- BEZ235 group and that of P70S6K(0.31±0.02) in the 1.00?M NVPBEZ235 group were both higher than that of the control group(0.10±0.02),the differences were significant(P<0.01, t=-6.26,-14.77); and compared with 0.50?M NVP-BEZ235 group, the protein levels of PI3 K, Akt and m TOR were significantly decreased in the 1.00?M NVP- BEZ235 group, the protein level of P70S6 K increased siginificantly, the differences were statistically significant(P<0.01, t=3.69,7.83,6.19,-11.55).Conclusion 1. The PI3K/AKt/m TOR signaling pathway participates in the development progress of hemangioma. 2. NVP-BEZ235 has shown the intervention effect on the hemangioma vasc?lar endothelial cells in vitro by the regr?lation of the PI3K/AKt/m TOR signaling pathway,and may have the dose dependent.