Molecular Mechanisms And Effects Of Modified Guizhi Fuling Pill On Human Ovarian Cancer Cell Caov-3

Objective To prove Molecular Mechanisms and effects of Modified Guizhi Fuling Pill on human ovarian cancer cell CAOV-3and study if VEGF,AKT,RAF,ERK1/2,IL-6and CD47are related with the Molecular Mechanisms.Methods The research is to study the effects on CAOV-3cells treated by Modified Guizhi Fuling Pill in vivo and vitro.Cell experiment:Cultivate the ovarian carcinoma cells with the culture medium including fetal calf serum. To observe the tumor inhibition rate by adopting MMT method and observe the effects on the CAOV-3cell apoptosis by adopting flow cytometry. At the same time, to observe the expression of VEGF,AKT,RAF and ERK1/2by adopting immunohistochemical method and to observe the expression of CD47. By adopting flow cytometry animal experiment:make the ovarian carcinoma cells into a nude mouses’shoulder blade and form the tumor. And then treat the nude mouses with Modified Guizhi Fuling Pill. From the day of the treatment, we observe the size of the tumor on alternate days and calculate the tumor inhibition rate. To detect the expression of the mRNA of VEGF、 AKT、RAF、ERK1/2. To observe the expression of VEGF and RAF by adopting immunohistochemical method and the expression of IL-6by adopting ELISE method.ResultCell experiment:(1)To detect The growth inhibition rate of CAOV-3after the treatment by the modified cassia twig tuckahoe pills on24h,48h,72h. The result shows the tumor cell survival rate decreased obviously (P<0.01) with the longer treatment and higher drug concentration, which indicated that the effective of the drug depends on the treatment duration and the drug concentration. Based on the inhibition rate, figure out IC50=0.9mg/ml. According to this indicator, we use the concentration of0,0.3,0.6,0.9,1.2mg/ml to do the cell experiment. (2) After the treatment on CAOV-3cells by Modified Guizhi Fuling Pill, the result shows The tumor apoptosis ratio rose with the increasing of the drug concentration, which indicates this drug can increase the tumor cell apoptosis (P<0.01). So the Modified Guizhi Fuling Pill can induce the apoptosis of the human ovarian cancer cells.(3)After24h of treatment,the expression of the mRNA of the VEGF,AKT,RAF and ERK1/2detected by RT-PCR is significantly decreased with the increasing therapeutic dose, which have dose-dependent (P<0.01). After24h of treatment, the expression of VEGF,AKT,RAF,ERK1/2is significantly decreased, which have Concentration-dependent (P<0.01)(4) The result of the flow cytometry shows the expression of the CD47in CAOV-3cell of the blank group is higher than the expression of the observation group (P<0.05), which indicates Modified Guizhi Fuling Pill can reduce the expression level of the CD47in human ovarian cancer cell.Animal experiment:(1)The effective on tumor growing by Modified Guizhi Fuling Pill:There is no difference among the groups before treatment(P>0.05);There is huge difference after treatment(P<0.01):PTV of the control group is8.75±0.73and the treatment group is2.98±0.52.The result indicates that Modified Guizhi Fuling Pill can restrain the growing of tumor.(2)The tumor inhibition rate is25.3%-53.0%by the Modified Guizhi Fuling Pill.(3)The result of the express of VEGF, AKT, RAF and ERK1/2:the control group has a high expression and there are some new expressions of vascular endothelial cells; the treatment group has a low expression.(4) Positive expression of VEGF and RAF protein were found in control group the positive expression rate is90%and87.5%in the control group and is lower in the treatment group (P<0.01).(5)The change of IL-6:the concentration of IL-6reduces gradually after3d,7d,11d and14d of the treatment in the treatment group, but the control group has no change.Conclusion(1) Modified Guizhi Fuling Pill can restrain the growth of the ovarian carcinoma cells.(2) Modified Guizhi Fuling Pill can make the cell cycle staying at the cycle of S, which can destroy the growth of ovarian carcinoma cells and induce cell apoptosis.(3) Modified Guizhi Fuling Pill may inhibit tumor angiogenesis and restrain the growth of the ovarian carcinoma cells by cutting VEGF-AKT signaling pathway.(4) Modified Guizhi Fuling Pill may restrain the growth, invasion and metastasis of the ovarian carcinoma cells by cutting MAPK signaling pathway.(5) Modified Guizhi Fuling Pill can reduce the expression of IL6.(6) Modified Guizhi Fuling Pill can reduce the expression of CD47. Possibly, it can destroy cancer cells by regulating the phagocytosis of the phagocytethe which is needed more research to support.