ABSTRACT
AIM: To study the effects of cladribine on growth and secretion activity of human umbilical vein endothelial cell line EA.hy926, and to investigate the mechanism of its anti-tumor effect by inhibiting endothelial cells. METHODS:The effects of cladribine at different concentrations on the cell viability were detected by CCK -8 assay.Apop-tosis and cell cycle distribution were examined by flow cytometry.The protein expression levels were determined by Western blot.The levels of tumor necrosis factor-α(TNF-α), transforming growth factor-β1(TGF-β1)and vascular endothelial growth factor(VEGF)secreted by EA.hy926 cells with cladribine treatment for 48 h were analyzed by ELISA.The nitric oxide(NO)production was measured by Gries method.RESULTS:Cladribine at 0.4~1 μmol/L inhibited the viability of EA.hy926 cells in time-and dose-dependent manners.The IC50was about 3.644 μmol/L.The results showed 43.74% cells in S phase when the concentration of cladribine was 0.4 μmol/L,and 77.23 % cells in S phase when the concentra-tion of cladribine was 1 μmol/L.The apoptosis was not induced by cladribine at 0.4~10 μmol/L.The protein expression of Bax and caspase-3 did not change.The expression of p21 increased and the p53 decreased(P<0.05).The levels of TNF-αand TGF-β1 secreted by EA.hy926 cells increased after cladribine treatment for 48 h.The levels of VEGF and NO decreased.CONCLUSION:Cladribine obviously inhibits the viability of EA.hy926 cells.The mechanism is related to the cell cycle arrest.Cladribine promotes the secretion of TNF-αand TGF-β1 by EA.hy926 cells and inhibits the secretion of VEGF and NO.
ABSTRACT
AIM:To explore the regulatory effect of chemokine CCL 3 on exosome secretion from human bone marrow mesenchymal stem cells(hBMSCs).METHODS: hBMSCs were stimulated with chemokine CCL 3 at different concentrations in vitro.The proliferation of hBMSCs was measured by CCK-8 assay and viable cell counting.Exosome se-cretion from hBMSCs was qualitatively analyzed by transmission electron microscope(TEM)and flow cytometry, and the quantitative analysis was carried out by flow cytometry and nanoparticle tracking analysis(NTA).RESULTS:Compared with control group,the viability of the hBMSCs detected by CCK-8 assay was increased when hBMSCs were treated with CCL3(P<0.05).The results of viable cell counting demonstrated that the number of hBMSCs was raised in CCL 3 group in a dose-dependent manner(P<0.05).The results of flow cytometry showed that hBMSCs expressed 3 CCL3-related spe-cific receptors,CCR1,CCR5 and CCR9.Compared with control group,the fluorescence intensity of CCR9 in CCL3 group was obviously enhanced.However,no significant difference of fluorescence intensity for CCR 5 and CCR1 was observed be-tween the 2 groups.The results of NTA demonstrated that the secretion capacity of CCL 3-induced hBMSCs was far less than that in control group(P<0.05).However, the microvesicles larger than 100 nm in CCL3 groups were increased(P<0.05).The above results indicated that the higher concentration of CCL 3 induced the lower secretion of exosomes.In addi-tion,the results of flow cytometry demonstrated that CCL 3-induced hBMSCs showed lower quantity of CD 9 +exosomes than those in control group(P<0.01).CONCLUSION:CCL3 promotes the proliferation of hBMSCs but depresses the secre-tion of exosomes in a dose-dependent manner.CCL3 affects the size distribution of exosomes and increases the number of nonfunctional microvesicles of larger than 100 nm in size.CCL3 induces the expression of CCR9 in hBMSCs.