| Diosmin is the aglycone of diosmetin(3',5',7'-trihydroxy-4'-methoxyflavone),which is plentiful in citrus fruit,rutaceae plants such as Radix zanthoxyli,madder plants such as Galium verum and other natural plants.In recent years it has been reported that diosmin can inhibit tumor cell proliferation,promote cancer cells with specific apoptosis and chemical prevention.Based on these fuctions,diosmin is considered to be potential anti-tumor drugs.In this paper,its effects on cell proliferation and apoptosis and potential targets in specific tumor cell have been studied.The main results are described as follows:(1)The antioxidant activities in vitro of four flavonoids with similar structure,including quercetin rutin hesperidin and diosmin were investigated by free radicalsscavenging assays(DPPH·,OH·,SOD,ABTS+·),ferrous ion chelating and total reducing power methods.By comparing with other three flavonoids,diosmin had strong antioxidant activity and its antioxidant ability was close to quercetin and rutin.(2)Diosmin on the proliferation inhibition of six cancer cell lines were screened by MTT method.It was found that diosmin showed the most effective in HepG2 and time dependent manner.Among the concentration 200~400 ?g/m L,its effect was significant and kept steady.Therefore,the concentration of 200 ?g/m L was selected to study the effect mechanism of diosmin on HepG2 cells.(3)Further study of the mechanism of diosmin on HepG2 cells showed the following results: The cells were observed by inverted microscope after 3~5 h incubation with diosmin-treated,after 7 h small granular substances appeared in cells and some cells became fragmented and losted with the emergence of dendritic crystals;According to HE staining,we found that the chromatin aggregation became confusing after 3~5 h incubation,after7 h cell membranes were damaged and most cells were shown fragmentation and small vesicles emerged;The cell cycle was detected by flow cytometry,which found that cells were significantly decreased in G0/G1 and G2/M phase;Cell apopotosis was detected by flow cytometry,and most of the cells were found in the late stage of apoptosis or death;According to DCFHDA we found that the fluorescence intensity was significantly increased after diosmin-treated,indicating that diosmin could increase the level of ROS in intracellular.HepG2 cells.Fluorescence microscopy also showed that the cells turned round from the original polygon and produce small round particles with different sizes and even the roles.Furthermore,cell fragmentation was serious as time prolonged.Therefore,we concluded that diosmin might induce the necrosis.(4)To search for the targets of diosmin on HepG2,2-DE was used to screen differental protein spots of the whole protein.In groups 976 protein spots were detected,among them 787 protein spots appeared on the control group,and 265 protein spots were found in diosmin-treated group.2 protein spots only appeard in the control group.Compared with the control group,there were 42 protein s in diosmin-treated group the expression increasing twice.7 differential protein spots were selected and analyzed by MALDI-TOF/TOF-MS mass spectrometry,and their biological functions were discussed according to Gene Ontology,KEGG Pathway and String Network.The possible action targets of diosmin-induced HepG2 cells apoptosis were investigated as follows: ACTA2 playing a key role in destroying the cytoskeleton structure and regulating the Raf/MEK/ERK/Ca D pathway and PI3K/Akt,ENO2 working in the biosynthesis of amino acids and the pathways of glucose metabolism.PKM developing the action in Ca signaling pathway and c AMP signaling pathway,Hsp A8 being involved in the regulation of TGF-?/RTK/clathrin,MKK4/7/JNK/c-JUN/Jun D or JNK/MKP/PTP/p38 and NF-?B pathway,and AFP participating in the regulation of Hippo-YAP and Akt pathway.Therefore,it was concluded that the damage of HepG2 hepatoma cells induced by diosmin may be a result of multipath regulation involving many relative proteins. |
PDF Full Text Request