Effects Of The Acetoacetate Extract Of The Vitex Negundo Seeds EVn-50 On Inhibition Of Growth And Induction Of Apoptosis In Human Retioblastoma Cells

ObjectiveTo screen the Acetoacetate extraction of Vitex Negundo Seeds against human Retioblastoma, and investigate the Acetoacetate extraction of Vitex Negundo Seeds (EVn-50) induce inhibition of growth induce apoptosis in RB cells, and evaluate the efficacy for Retioblastoma therapy by EVn-50 in vitro, in order to provide a laboratory and theoretic evidence for searching for a new potent and promising candidate for Retioblastoma chemotherapy.MethodsHuman retioblastoma cells were cultured in vitro. Then, MTT assay was used to determine the effect of the Acetoacetate extraction of Vitex Negundo Seed (EVn-50) on the proliferation of RB cells. The inhibition of the growth by EVn-50 in RB cells was used to test by the cell counting after trypan blue staining. Colony formation assay in soft agar was used to detect the colony formation inhibitory effect of on RB cells. Flow cytometry (FCM) using PI staining was used to analyse the apoptosis rate treated with EVn-50 in RB cells. Fluorescence after AO/EB staining was used to observe the morphologic changes of apoptosis induced by EVn-50. DNA agarose gel electrophoresis was used to confirm apotosis induced by EVn-50 in RB cells.ResultsThe MTT assay showed that the acetoacetate of extractions from Vitex Negundo Seeds inhibited the cell viability of RB cells to certain degrees. EVn-50 could be considered as the effective extract of Vitex Negundo Seeds, with IC50 value was 9.60μg/mL in RB cells. Furthermore, the cell counting data had shown that the EVn-50 inhibited the growth of RB cells in a time-and dose-dependent manner. Colony formation assay in soft agar showed that EVn-50 significantly inhibited the non-anchorage dependent growth of RB cells in a concentration-dependent manner. Flow cytometry(FCM) analysis after PI stainning indicated that the apoptosis rate of RB cells was (2.4±0.1)%,(17.4±0.6)%,(40.7±0.7)% respectively after treatment with EVn-50 at the concentrations of 0.3, 3.0 and 30.0μg/ml for 48h. Meanwhile, the apoptosis rate of RB cells was (20.8±0.4)%,(40.7±0.7)%,(63.1±0.8)% respectively after treatment with EVn-50 at the concentrations of 30.0μg/ml for 24h, 48h and 72h. In comparision, they were much higher than the apoptosis rate ( 0.8±0.3)% in control group, which revealed that EVn-50 significantly induced apoptosis in a time- and dose- dependent manner. Typical morphologic changes of apoptosis in RB cell could be observed after treatment with EVn-50 by fluorescence microscope using AO/EB fluorescence staining. DNA agarose gel electrophoresis shown that DNA ladder bands could appear after treatment with EVn-50 at 0.3μg/mL,3.0μg/ml,30.0μg/ml respectively for 48h as well as EVn-50 at 30.0μg/ml for 0h,24h,48h,72h.Conclusion1. EVn-50 can inhibit the proliferation and growth of RB cells, in a time- and dose- dependent manner. It is the effective extract of Vitex Negundo Seeds against human Retioblastoma.2. EVn-50 can significantly induce apoptosis of RB cells, in a time- and dose- dependent manner.