The Inhibition Effects Of2-deoxy-d-glucose On The Growth Of Human Umbilical Vein Endothelial Cells And Hemangioma

BackgroundHemangioma is the most common benign tumor in infants,60%of which are locatedat head and neck and most of which is on the skin suface.In consequence, hemangiomaseriously affects the outlook of the patients, amounts enormous psychological pressure tothe patients, affects the physiological function of patients,and even threats patients’ lifedue to severe operative complications such as bleeding, ulcer, infection or local oppression.The exact pathogenesis of hemangioma have yet remained unclear, there is no efficienttreatment for all types of hemangioma, so it is of clinical significance to find an efficientteeatment for hemangioma.2-deoxy-D-glucose(2-DG) is an analogue of glucose, it can intervene in several keysteps in tumor growth through inhibiting glycosis and N-linked glycosylation ofendothelial cells and inducing cell apoptosis.ObjectiveWe intend to study the effects of2-DG in the treatment of hemangioma through thein vivo and in vitro experiments. In vivo experiments were designed to explore the effectof2-DG on the human umbilical vein endothelial cells proliferation, migration, tube formation and cell apoptosis, from which we can get an insight into a possible mechanismof how2-DG affects the endothelial cells of hemangioma; Through the studies onhemangioma formation, structure and cell morphology in nude mice, we intend tounderstand the possible effects of2-DG on transplanted tumours.Methods1. Four methyl thiazolyl tetrazolium colorimetric assay.The HUVECs were cultured in96well culture plate, ECM containing0mM,0.06mM,0.6mM,6mM,9mM2-DGrespectively were added in the control group and experimental group.On the second day,the third day and the fourth day,absorbance value was acquired by enzymeimmunoassay instrument.2. The inhibitory effect of2-DG on the HUVECs growth and morphology. The cells wereseeded in6-well plate. ECM containing0mM,0.06mM,0.6mM,6mM,9mM2-DGrespectively were added in the control group and experimental group. At0h,24h,48hthe numbers and morphological changes of HUVECs were observed by OLYMPUSinverted microscope.3. Scratch assay. The cells were seeded in6-well plate.2hours after20μL mitomycin Cwas added cross line were made per plate cell. ECM containing0mM,0.06mM,0.6mM,6mM,9mM2-DG respectively were added in the control group and experimental group.At0h,12h the effect of2-DG on HUVECs migration were observed by OLYMPUSinverted microscope.4. Matrigel tube formation assay.50μL Matrigel evenly added to96-well plate, HUVECswere seeded on it after the Matrigel solidified. ECM containing0mM,0.06mM,0.6mM,6mM,9mM2-DG respectively were added in the control group and experimentalgroups. At0h,12h the effect of2-DG on HUVECs tube formation were observed byOLYMPUS inverted microscope.5. Flow cytometry for the effect of2-DG on HUVECs apoptosis. The cells were seededin6-well plate after digestion. ECM containing0mM,0.06mM,0.6mM,6mM,9mM2-DG respectively were added in the control group and experimental group, cultured for12hours.After pretreatment5μL FITC Annexin V and5μ L PI were added respectively, then400μL Bing of Buffer were also added, the effect of2-DG onHUVECs apoptosis were detected by using flow cytometry.6. Establishment of the hemangioma model in nude mice. The fresh infantile hemangiomaspecimens were cut into0.8cm X0.6cm X0.6cm blocks, and transplanted into nudemice subcutaneously, two blocks for each nude mice. Transplantation work was completedin2hours. Finally, according to the principle of randomization the nude mice were dividedinto control group and experimental group.7. Transplanted tumour volume observation. Observation regularly of the living conditionof nude mice and the growth of transplanted tumors, The volume of tumour wasobserved and recorded on a regular basis from the first day to the35thday postoperation.8. Histopathological observation. On the35th day after the tumour transplantation, thetumors were removed.HE staining was done for all the remouved tumors.Tissue structure and cell morphology were observed at a magnification of400×underthe optical microscope.9. CD31Immunohistochemistry and analysis. On the35th day after the transplanted tumorwere removed, Immunology was done according to the manufacturers’ manuals. Thetransplanted tumor sections were observed under optical microscope at400timesand the number of positive cells were counted to calculate the index of positive cells.ResultsThe inhibitory effects of2-DG on HUVECs tube formation, migration and inductionof cell apoptosis was observed after12hours of treatment. HUVECs can hardly form thecavity structure in the6mM2-DG treatment group; In the cell migration experiment,HUVECs filled the space of scratch area in the control group, while the cell migration ratedid not exceed50%in the6mM2-DG treatment group; Flow cytometry showed theapoptosis rate was10.63%in the control group, while21.77%and25.07%of apoptosisrate were observed in6mM and9mM2-DG treatment groups, respectively. At24h and48h after2-DG treatment, in the cell morphology experiment the number of HUVECs inthe control group were continuously increased, while the cell numbers in experimental group decreased at different degrees. The cell morphology also changed, The cell sizesbecame smaller with shrunk cytoplasma. In MTT assay72h treatment with0.6mM2-DGsignificantly inhibited cell viability comparing to control group, and the inhibitory effectswere more obvious in the6mM and9mM2-DG treatment groups each with more than60%inhibition. In in vitro experiments,the tumor volume in control group showed agradually increasing trend, while the tumor volume in the experimental groups decreasedsteadily.Under light microscope,the capillary densities in tissue implants of experimentalgroups were significantly decreased relative to that of control group.Fibrotic tissues andfatty infiltration were also observed in treatment groups.ConclusionThis study showed that2-DG significantly inhibits HUVECs proliferation, migrationand tube formation，significantly alters cell morphology, induces cell apoptosis,all in doesdependent manner; In vivo2-DG treatment caused obvious reduction of microvasculature,fibrous tissue proliferation with fat infiltration.2-DG demonstrats the inhibitory effect onendothelial cells both in vitro and in vivo, providing us with a new idea for the efficienttreatment of hemangioma.