Study On Inhibitory Effect And Mechanism Of PESV On Human Ovarian Cancer Cell Line SKOV3

Ovarian cancer is a common malignant tumor of the female genital organs. In recentyears, the morbidity and mortality of ovarian cancer have a slow upward trend. Accordingto the latest statistics, its incidence rose from third to second place in gynecologic cancer.Due to the majority of the patient are clinical late stage already, the mortality rate is thehighest of the various types of gynecologic oncology. Currently, the standard regimen forovarian malignancy is the Tumor cytoreductive surgery and platinum-based adjuvantchemotherapy, but the5-year survival rate is still hovering at about20%～25%, there isstill a serious threat to the life of women. How to control the development of ovariancancer and to find new treatments, have become a key research project.The premise of tumor proliferation, migration is angiogenesis. Therefore, inhibittumor angiogenesis has great significance for controlling of tumor progression, improvingprognosis and survival rate of patients. Chinese medicine treatment tumor diseases have along history, Scorpion venom has been used in the treatment of many diseases as atraditional Chinese medicine, and its effect of anti-tumor also has draw attention.Polypeptide extract from scorpion venom (PESV) is a polypeptide extracted fromScorpionidae. The early experiments of this study group confirmed that PESV have a goodanti-tumor effect in variety of tumors such as Liver cancer, non-small cell lung cancer andso on. However, the mechanism of PESV effect on ovarian cancer has not been elucidated.So our study group explore the possible mechanism of PESV inhibits human ovariancancer by relational detection methods. ObjectiveThis study was to observe the effects of PESV on the cell proliferation, cell cycle andexpression of VEGF, TSP-1, and Id-1in ovarian cancer cell line SKOV3, to investigate thepossible mechanism of PESV anti-tumor effect, and to provide a new theoretical basis forPESV anti-tumor effects.Methods1. Observation of cellular morphology under microscopy and detection the effect of PESVon proliferation activity in SKOV3cells by MTT assay．2. Detection the effect of PESV on cell cycle distribution in SKOV3cells by flowcytometry.3. Immunocytochemistry and western bolt were applied to detect the effects of PESV onthe protein expression of VEGF、TSP-1and Id-1.4. RT-PCR were applied to detect the effects of PESV on the mRNA expression of VEGF、TSP-1and Id-1.Results1. Effect of PESV on morphology of SKOV3cellCell morphology of the control group showed that: short uniform，polygonal，uniformsize, translucent cytoplasm,well adherence, vigorous growth. After treatment with PESV,contrasting to control group, density of cells was decreased, cells became round and grainyenhanced intracellular, cells shedding and floating. With the PESV concentration increased,the phenomenon is more pronounced.2. Effect of PESV on proliferation of SKOV3cellsPESV remarkably inhibited the proliferation of SKOV3at the range of12.5～200μg/mL, the inhibition gradually strengthen with the increase of the concentration.When the concentration of PESV were50、100、200μg/mL,the growth inhibition ratiowere38.78%、53.30%、79.22%respectively. PESV has a significant inhibitory effect oncell proliferation in a dose-dependent. The differences between the groups were statistically significant (P<0.01).3. Effect of PESV on cell cycle of SKOV3cellsWith the increase concentration of PESV,cell proportion of G0/G1phase wasgradually increased from45.80%±1.05to69.28%±1.03, S phase decreased from32.50%±0.98to17.23%±0.31, There was significant difference between different groups(P＜0.01).The effect of PESV on cell cycle in a dose-dependent.4. Effect of PESV on protein expression of VEGF、TSP-1、Id-1of SKOV3cell4.1immunocytochemistry After treatment with PESV48h, the expression levels ofVEGF、Id-1were significantly decreased compared with that of the control group (p<0.01).There was a positive correlation between the expression of VEGF and Id-1,r=0.819,P<0.01.TSP-1was unregulated by PESV,150μg/mL group was strongly positive(P<0.01),100μg/mL group was positive (P<0.05), The difference between50μg/mL groupand control group was not significant.4.2western blot The expression of VEGF in PESV100,150μg/mL group wasdecreased (P<0.01), Id-1were significantly reduced in all dose group (P<0.01).PESVexhibited a dose dependent up regulatory effect on the expression of TSP-1,150μg/mLwas significantly enhanced (P<0.01). Statistical analysis, the expression of Id-1and VEGFwere positively correlated, r=0.762, P <0.01, while Id-1and TSP-1was negativelycorrelated，r=-0.741, P<0.01.5. Effect of PESV on mRNA expression of VEGF、TSP-1、Id-1of SKOV3cellPESV treatment can down regulate the VEGF、Id-1mRNA, at the same time themRNA expression of TSP-1are up-regulated. Compared with the control group, VEGFand Id-1mRNA expression of PESV150μg/mL were significantly reduced (P <0.05),TSP-1mRNA expression was significantly increased (P<0.05).ConclusionsPESV can inhibit the proliferation on human ovarian cancer SKOV3cells significantly,and arrest the cells stay in G0/G1.In addition, PESV also can affect protein and mRNA expression of VEGF、TSP-1、Id-1. Anti-tumor effects of PESV may be carried out byinhibiting the tumor cell proliferation, affecting cell cycle distribution and impacting theexpression of key factors in the process of tumor angiogenesis.