Effect Of Epinodosin, An Ent-kaurane Diterpene, On Growth Inhibition,Differentiation Induction And Cytoskeleton In HL-60 Cell

In this study, the effects of epinodosin, a kind of ent-kaurane diterpenoids, on the growth, cellular morphology, NBT-reducing ability, cellular uptake, cell ROS and cell surface markers CD11 b of HL-60 cells were determined by trypan blue exclusion method, giemsa stainning and flow cytometry. In addtion, the effects of epinodosin on cytoskeleton of HL-60 cells during the process of differentiation induction were detected by immunofluorescence assay. The results as follows:1.The results of MTT and trypan blue staining method showed that 2.0 ?M, 4.0 ?M,6.0 ?M and 8.0 ?M epinodosin had a dose- and time-dependent growth inhibition effect on HL-60 cells. 6.0 ?M epinodosin and 1.2 ?M ATRA treatment group had same results.2. The results of PI staining flow cytometry indicated that epinodosin could result in S phase in HL-60 cells and inhibit cell growth in a dose-dependent manner.3.The results of giemsa stainning, NBT-reducing ability, cellular uptake and flow cytometry showed that metamyelocytes and lobate nucleus increased, the ratio of nuclear/cytoplasm decreased; NBT-reducing ability, cellular uptake and the expression of CD11 b of HL-60 cells enhanced markedly in a obvious dose-dependent manner. These indicated that differentiation of human HL-60 cells into granulocyte could be induced by epinodosin.4. The results of DCFH-DA staining and flow cytometry indicated that the level of ROS increased obviously in cells treated with 4.0 ?M and 8.0 ?M epinodosin for 6 h;the whole level of ROS in cells treated for 12 h and 24 h was lower than in cells treated for 6 h, but a little higher that control group, these indicated that epinodosin could result in the increasing of ROS in a short period. After 0.3 mM NAC and 8.0?M epinodosin were added and cultured for 6 h, the level of ROS in cells was lower than in cells treated with 8.0 ?M epinodosin alone, the results indicated that NAC could decrease the produce of ROS in cells caused by epinodosin.5. By detection the expression of CD11 b in cells after the combination treatment with 0.2 mM APO and 8.0 ?M epinodosin for 72 h, CD11 b positive cell rate incombination treatment group was 33.59% which lower than cells treated with 8.0 ?M epinodosin alone, this indicated that APO could inhibit the differentiation induction caused by epinodosin, namely, the produce of ROS in the process of cell differentiation was closely related to NADPH oxidase.6. By detection the expression of CD11 b in cells after the combination treatment with 0.1 ?? DPI and 8.0 ?M epinodosin for 72 h, CD11 b positive cell rate in combination treatment group was 26.44% which lower than cells treated with 8.0 ?M epinodosin alone, this indicated that 0.1 ?? DPI could decrease the differentiation induction caused by epinodosin, namely, the produce of ROS in the process of cell differentiation was closely related to NADPH oxidase.7.The results of rhodamine-conjugated phalloidin staining showed that cells treated with 6.0 ?M epinodosin for 24 h presented obvious fluorescence spots and microfilaments became dense; as compared with 24 h, the whole level of fluorescence in cells treated with 6.0 ?M epinodosin for 72 h decreased, a fluorescence ring distributed along the cell peripheral, and a fluorescence spot gathered at one side of cell. All these indicated epinodosin could change microfilaments in HL-60 cells.8.The results of immunofluorescence assay for microtubule showed that microtubules in cells after the treatment with 6.0 ?M epinodosin for 24 h became more dense than control group; as compared with 24 h, cells after treament with 6.0?M epinodosin for 72 h presented a bright fluorescence ring along the cell peripheral,the density of microtubules became thin. The results showed that epinodosin could result in the assembly of microtubules in the process of cell differention.9.The results of immunofluorescence assay for vimentin showed that a bright fluorescence spot appeared in cells treated with 6.0 ?M epinodosin for 24 h; after treated for 72 h, the fluorescence spot appeared in more cells, bundle filaments became thick. From all, we think epinodosin could induce the assembly of vimentin in the process of cell differention.