The Study Of Weisiensin B,an Ent-kaurane Diterpene, On Growth Inhibition And Differentiation Induction In Human Giloma Cell Line U87

Glioma is the most common primary brain tumor. The effects of curing glioma treatment by surgery, chemotherapy and radiotherapy are not obvious, so it is necessary to find a more effective way of treatment. Inducing differentiation therapy is a new treatment of cancer therapy, by which tumor cells differentiate to or close to normal cell without killing the cells. In addition, the new therapy has several advantages such as a small dosage, low toxic side-effects. Therefore, looking for inducing differentiation agent with efficiency and low toxicity plays an important role in the treatment of glioma.Isodon plants, the folk traditional herbe, has a variety of pharmacological effects, such as anti-bacterial, anti-tumor and anti-bacterial. As one of kinds of compounds isolated from Isodon plants, ent-kaurane diterpenoids, the content of which is rich, are the main secondary metabolites. The study showed that the compound with varieties of physiological activity has the great potential in anti-tumor. Weisiensin B belongs to ent-kaurane diterpenoids isolated from Rabdosia weisiensis. Studies have suggested that the compound has a strong cytotoxicity, but the mechanism of its anti-cancer activity is still not clear, needing the further study. At present, the study about the effect of Weisiensin B on U87 cells differentiation has not been reported in the literature.In the study we used the ent-kaurane diterpenoids Weisiensin B as subjects drugs, and the human brain glioma U87 cells as the subjects. Trypan blue exclusion and MTT method are used to detect the Weisiensin B on U87 cell growth inhibition, using flow cytometry to detect the effects of compounds on U87 cell cycle distribution; Microscope observation and Giemsa staining method are used to study the effect of Weisiensin B on U87 cell morphology; Using indirect immunofluorescence and fluorescent staining observed the effects of compounds on the GFAP and Vimentin content in cells and microfilament, microtubule cytoskeleton rearrangement. The main results as follows:(1)Trypan blue staining and MTT method results showed that the Weisiensin B has obvious growth inhibition on U87 cell, as the enhancement of drug concentration, cell growth inhibition was increased,showing the concentration and time dependence.(2) The inverted microscope observed the, Giemsa staining method observed the cellular morphological changes, the results showed that cell morphology have been changed after Weisiensin B affecting to U87 cell, the control cells presented short spindle, both sides had short projections, the nucleus were bigger. After being treated with different concentrations of compounds, cell volume was larger, nuclei was smaller, nucleus mass ratio decreased, and protuberance swelled. This phenomenon in high concentration treatment group is more obvious.(3)The flow cytometry instrument detected cell cycle distribution after the PI staining, the results showed that different concentration Weisiensin B has influence on the U87 cell cycle distribution in 48 h and 72 h, can cause G0/G1 phase cycle arrest of U87 cell, and have concentration dependence, appearance of apoptotic peak was not detected. AO/EB double staining results also showed that within the scope of the drug concentration did not cause apoptosis.(4)Scratch method observation U87 cell migration results showed that Weisiensin B had strong inhibition effect to U87 cell migration. With the concentration increase and the time extension, Inhibition effect was more obvious,showing the time dose effect.(5)We investigated the concent chage of GFAP and Vimentin in U87 cell by immunofluorescence and flow cytometry,the results showed that after treatment with Weisiensin B can cause the content increase of GFAP and decrease the Vimentin content in U87 cell,and in a concentration dependent manner.therefore,Weisiensin B can induce U87 cell differentiated toward mature astrocytes.(6)Rhodamine-labeled phalloidin staining and immunofluorescence staining observe showed that Weisiensin B caused U87 cell microfilament skeletal depolymerization in a concentration dependent manner. Weisiensin B can affect microtubule cytoskeleton rearrangements on U87 cell,in the control group, microtubule is mainly distribute in the cell periphery and the less intracellular,after treatment with Weisiensin B, microtubule toward perinuclear aggregation and microtubule increase inside the cell.The results show that Weisiensin B can induce U87 cell skeleton change,which may be related to the changes in cell morphology during cell differentiation.(7)After DCFH-DA staining use Flow cytometry assay and fluorescence microscopy observations intracellular ROS content change,the result suggest that the U87 intracellular ROS were significant increase dependent by time-dose when cells were treated by Weisiensin B.In conclusion, Weisiensin B could induce U87 cell morphological change,strong G0/G1 phase arrest and causing growth inhibition.further study found that compound can increasing the intracellular content of GFAP and decreased the content of Vimentin induce the cell differentiation toward normal glial. Weisiensin B could cause intracellular microtubule rearrangements and microfilament depolymerization.The study is the first to investigated the influence of Weisiensin B induce differentiation for U87 cell,prove a basis for further proved the anticancer mechanism of Weisiensin B.