Experimental Study Of Endostar’s Effect On Tumor Vessels Angiogenesis And Normalization

Objective Because of its early pelvic and abdominal cavity planting metastasis,lymph node metastasis,clinical symptom not obvious, the pathogenesis of complex, high malignant degree, the mortality of ovarian cancer is the highest of all gynecological tumors. It has been severely threatening the health and life of women.The standard treatments contain surgery,radiation and chemotherapy, but the recurrence rate is as high as 60%.The majority of recurrent patients is resistant of the first-line chemotherapy drugs-cisplatin, eventually leading to a poor prognosis.So it is necessary to find a new and effective treatment method, comprehensively improve the overall survival rate of patients with ovarian cancer, and improve the patient’s quality of life. So it is necessary to find a new and effective treatment method, improving the overall survival rate of patients with ovarian cancer and the patient’s quality of life comprehensively.As the other malignant tumors, the growth of ovarian cancer is dependent on angiogenesis. The growth of tumor needs nutrients and oxygen which are provided by angiogenesis, at the same time,angiogenesis could delivery tumor cells nearby and in distance,leading to tumor metastasis.If we can find a method which can effectively prevent the formation of new blood vessels,we may achieve the goal of inhibiting tumor growth and metastasis. Tumor vascular structural abnormalities can also lead to abnormal blood vessel function. Incomplete tumor blood vessels lead to blood disorders, ineffective circulation increase, the formation of the inequality of the tumor blood flow in time and space, and changes of tumor hemodynamic directly influence the metabolism of tumor microenvironment, mainly for hypoxia and acidosis. Low pH and acidosis are of reciprocal causation and intensifying, leading to reduced transport chemotherapy drugs and radiation resistance. Can we development a new treatment which may make abnormal tumor vessels return to normal ones and furthermore delivery oxygen and drugs effectively?Endostar is a new type of soluble vascular endothelial cells inhibitors, which can selectively inhibit endothelial cell proliferation, migration, and angiogenesis.Previous researches have demonstrated that anti-angiogenesis drugs can make the normalization of tumor blood vessels transiently.This study was divided into two parts.Part one is observing the affect of Endostar on proliferation, migration and tube formation on HUVECs that were induced by supernatant fluid of SKOV3/DDP cells,to explore the influence of Endostar on angiogenesis in ovarian cancer.Part two is to observe an "normalization"time window on the successful orthotopic transplantation nude mice model.Methods Part one:The preparation of supernate of SKOV3/DDP cells:Cultivating SKOV3/DDP cells, when the cell confluence was 70%～80%,to change in serum-free medium RPMI-1640, continuing to cultivate 24h, centrifugal to collecte supernatant, with the RPMI-1640 medium containing 10% FBS 4:1 mixed. Set aside. MTT method was used to observe the effect of Endostar on proliferation of HUVECs that were induced by supernatant fluid of SKOV3/DDP cells.The concentrations of Endostar were 10-7,10-6,10-5,10-4,10-3,10-2 and 10-1mg/ml and the observation time points were 24h,48h and 72h. Wound healing assay was used to observe the effect of Endostar on migration of HUVECs induced by supernatant fluid of SKOV3/DDP cells. Cells were seeded into 6-well plates.When the cell confluence was 90%,vertical scratching the plate with a tip of 20μl and washing floating cells with 1 x PBS. The final concentrations were 10-6,10-4,10-2mg/ml, respectively.After incubated for another 4h with serum-free medium RMPI-1640,pictures were taken under an inverted phase contrast microscope.Migration mobility was calculated by the distance of the cells spacing that was measured by ImageJ. The difference between the three groups was measured by drawing diagrams and using SPSS18.0 statistical analysis.Tube formation was used to observe the effect of Endostar on angiogenesis of HUVECs induced by supernatant fluid of SKOV3/DDP cells. Spreading Matrigel gel into 96-well plates when it becames liqiud state.Each hole was 50μl and then put the plate into incubator under 37℃ for 30minites until freezing. HUVEC cells in logarithmic growth phase were harvested by trypsin digestion and added into the gel spread holes at a density of 1×105L/ml. Control groups were also included. The final concentrations of10-6,10-4,10-2mg/ml, respectively. After 4h of culture,we took out the plates and took photos to observe the tube formation under inverted microscope.We used ImageJ software to measure the length of tubes and calculated HUVECs tube formation rate under different Endostar concentrations. The difference between the groups was measured by drawing diagrams and using SPSS 18.0 statistical analysis.Part two:MTT assay was used to determine the DDP resistance of SKOV3/DDP cells. DDP at the final concentrations were 0.1,0.2,0.4,0.8,1.6,3.2,6.4, and 12.8 μg/mL, respectively. After 48h of culture, the absorbance value at 490nm was detected on a microplate reader. The data were recorded and drown into an survival and growth curve of different DDP concentrations.SKOV3/DDP cells were used to establish orthotopic tumor model in nude mice. SKOV3/DDP cells were adjusted to appropriate density as single cell suspension, which were injected subcutaneously at the neck of each nude mouse. When the tumor grew into>l cm in diameter, the animals were sacrificed by cervical dislocation, and the tumors were isolated under sterile conditions, freshly cut into small pieces, and inoculated into the left ovary in mouse. After tumor implantation, the animals were under close monitoring and observed the progress of the tumors.When abdominal palpation touched the mass clearly, the mice were sacrificed. The tumor lump was isolated, fixed, cut into sections, and stained with hematoxylin and eosin (HE). The morphologies of normal ovarian tissue and orthotopic and subcutaneous tumors were compared.After all models established,a total of 30 satisfactory mice were randomly divided into 6 groups with 5 mice in each group. Among them,3 groups were injected daily with 0.2 mL Endostar (10 mg/kg) intraperitoneally (i.p.) and sacrificed respectively 3,6, and 9 days after the injection; the other 3 control groups were i.p. injected daily with 0.2 mL saline and sacrificed respectively 3,6, and 9 days after the injection. The integrity of tumor, vascular endothelial cells at different time points after Endostar treatment was measured by immunofluorescent staining of CD31.The ImageJ software was used to count the complete vessels.The concept of countably complete vessels was that the endothelial cells were dyed by CD31 antibody and formed a integrated.Result Part one:That cells were dealt with respectively after 24h,48h,72h with different concentrations (10-7,10-6,10-5,10-4,10-3,10-2,10-1mg/ml) of Endostar had no obvious effect in HUVEC cells proliferation. There was no ststistics differences among groups. The Endosar administration team had wider space than the matched group,two of which had statistics differences when they were compared (P< 0.05).But the HUVEC cells migration effects on different concentrations had no obvious differences,which has no statiscally significant (P>0.05). The tubes came into being in the matched group and the experimental group,were distributed uniformly,had no obvious difference under the microscope. After using ImageJ picture analysis software to calculate the length of tubes,the difference between the matched group and the experiment group has no statistical significance (P>0.05)Part two:The IC50 of DDP on SKOV3-DDP and SKOV3 cells were 11.647±1.078 and 2.486±0.983 μg/mL respectively. Compared with the sensitive SKOV3 cell line, the drug-resistant SKOV3-DDP cells were more resistant to DDP, and the difference was statistically significant (F=2.386, P<0.05).After dissection, a mass was visible in the left ovary area, which was with retroperitoneal adhesions. However, the orthotopic tumor masses showed nuclei with greater nuclear-cytoplasmic (N/C) ratio which was matched with the charater of malignant cells.The tumor vasculatures in saline control groups were irregular and tortuous, with varied diameter and multiple branches, the vast majority of which were covered with incomplete endothelial cells. However,3 and 9 days of Endostar administration improved the vascular integrity (23±4.58% and 17.67±8.34%, P>0.05) and reduced the number of vascular branches. Especially, the tumor vasculatures in mice treated with Endostar for 6 days showed complete vascular wall, uniform diameter, and significantly improved integrity (75.33±8.02%, P<0.01 vs. 3 and 9 days respectively).Conclusion Part one:Endostar had no obvious effect on proliferation and tube formation of HUVEC cells induced by supernate of SKOV3/DDP cells and little effect on HUVEC cells migration.Part two:SKOV3/DDP cells were used to establish orthotopic tumor model in nude mice successfully. On the basis of the models,we observed that Endostar treatment produced a short "time window" of normalization in tumor vasculature, i.e.,6 days of Endostar administration resulted in a nearly normalized tumor vasculature.