Study On The Synergetic Inhibition Of SKOV3 Growth By Bevacizumab And Cisplatin

Ovaries have more complicated features such as embryonic development,tissue anatomy,and endocrine function,and they are placed deep in the pelvic cavity,leading to early occurrence of local or even systemic symptoms of ovarian cancer.The incidence is subtle.Due to the lack of obvious clinical symptoms in the early stage of ovarian cancer,about 70%of patients have been diagnosed at the advanced stage,often accompanied by peritoneal metastasis or malignant ascites.At present,the main strategy for the treatment of ovarian cancer is to perform cytoreductive surgery to the greatest extent,and to give postoperative chemotherapy combined with paclitaxel and platinum drugs.Cisplatin and its analogue carboplatin have been widely used as first-line chemotherapy drugs in the treatment of ovarian cancer.However,the majority of patients relapse within about 2 years after combined treatment and are often accompanied by platinum-resistance,resulting in a 5-year survival rate of patients with advanced ovarian cancer still hovering around 30%^[1].Cisplatin is an inorganic metal-platinum complex,and its drug activity is negatively correlated with the concentration of chloride ions.When it enters the body’s low-chlorine cells,the activity increases,and hydration dissociation will soon occur,resulting in a positively charged hydration complex ion.Under the electrostatic attraction of DNA,the hydrated complex ions migrate toward the cell nucleus,eventually forming an adduct and changing the normal DNA replication,leading to DNA replication disorder of the tumor cells,thereby inhibiting the division of tumor cells^[2].The drug concentration of cisplatin determines its anti-tumor activity,that is,the higher the concentration of each dose in a short period of time,the better the anti-tumor efficacy^[3].Because cisplatin itself limits the use of large doses of human organs such as the ears,kidneys,and digestive tract,it affects the killing effect on tumor cells.In addition,due to the recurrence of ovarian cancer patients and the prolonged treatment time,the occurrence of platinum resistance phenomenon also limits its clinical use.Currently in the treatment of ovarian cancer more combined medication and change the route of administration to achieve the purpose of attenuated^[3].With the in-depth study of the molecular biology mechanisms related to the development of ovarian cancer,new therapeutic targets for ovarian cancer have been continuously discovered,especially vascular targeted therapy.Bevacizumab is a recombinant humanized monoclonal antibody that can inhibit the binding of VEGF to its receptor,neutralize the biological activity of VEGF,block the normal conduction of multiple signaling pathways downstream of VEGF,and destroy and suppress the supply of cancer cells.Nutritional neovascularization slows tumor growth and distant metastasis to achieve therapeutic goals^[4].In addition,bevacizumab can normalize the blood vessels of tumors and help chemotherapeutic drugs enter the tumor body,thereby exerting more effective anti-tumor effects.The use of bevacizumab in combination with capecitabine and cisplatin has been approved for the clinical treatment of advanced gastric cancer.Studies have shown that combined with bevacizumab significantly increases the patient’s progression-free survival compared with chemotherapy alone^[5].The efficacy of bevacizumab combined with cisplatin in the treatment of malignant pleural effusion of non-small cell lung cancer was significantly higher than that of the cisplatin alone group.It was also found that the combination of bevacizumab and cisplatin administered in patients with advanced malignant melanoma showed a short-term efficacy that was superior to that of cisplatin alone.This article aims to study the mechanism of the combination of VEGF-targeted drug bevacizumab and commonly used chemotherapeutic drug cisplatin on SKOV3 cells,providing some relevant theoretical support for the treatment of ovarian cancer.ObjectiveTo detect the expression of VEGF and MMP-9 in normal,benign,and malignant ovarian epithelial tissues,and to analyze the correlation between the expression levels of these two proteins in ovarian cancer tissues.And focused on the mechanism of bevacizumab in combination with cisplatin to inhibit the growth of SKOV3 cells.Materials and methods1.Subjects:41 patients with ovarian epithelial malignancies（ovarian cancer group）were recruited.No radiochemotherapy was performed before the operation;30patients with benign ovarian tumors（group with benign ovarian tumors）;30 patients with benign uterine lesions who underwent ovariectomy（Normal control group）.All of the above patients were diagnosed by pathology.There was no significant difference in age between ovarian cancer group,ovarian benign tumor group and normal control group（P>0.05）.2.Methods:The expression of VEGF and MMP-9 protein in ovarian tissue was detected by immunohistochemistry in 101 cases of ovarian epithelial tissues;MTT assay was used to detect bevacizumab and bevacizumab combined with cisplatin in ovarian cancer cells SKOV3.Inhibition of proliferation in vitro;Transwell assay to detect the effects of bevacizumab and cisplatin alone and in combination on the invasion of SKOV3 cells;Annexin V-FITC and PI double staining combined with flow cytometry to detect bevacizumab and cisplatin alone and the effect of combined use on the apoptosis of ovarian cancer cells;Western blot was used to detect the effect of bevacizumab combined with cisplatin on the expression of key proteins,MMP-9protein and Bad protein in VEGF downstream signaling pathway.3.Statistical Methods:SPSS 19.0 was used for statistical analysis.The positive rate of VEGF and MMP-9 protein expression in ovarian cancer group,ovarian benign tumor group,and normal control group were tested using multi-sample 2 test.Kruskal-Wallis H non-parametric test was used for immunohistochemistry;VEGF and MMP were detected in ovarian cancer tissues.Pearson correlation analysis was used to correlate the expression of-9 protein;the effect of bevacizumab alone and in combination with cisplatin on the invasion and apoptosis of SKOV3 was analyzed by One-way analysis,and the test level wasα=0.05.Results1.The expression of VEGF and MMP-9 protein in three groups of ovarian tissues.The results of immunohistochemistry showed that statistical analysis of VEGF and MMP9 protein expression and immunohistochemistry scores showed that VEGF and MMP-9 were highly expressed in ovarian cancer tissues（P<0.001,vs normal control group）.In the ovarian benign tumor group,Pearson’s correlation analysis showed thatχ2=6.034,r=0.360,P=0.014,that is,VEGF and MMP-9were positively correlated in the ovarian cancer group.2.Inhibitory effects of bevacizumab and bevacizumab combined with cisplatin on proliferation of ovarian cancer cells in vitro.The ovarian cancer cells SKOV3were treated with bevacizumab at different concentrations（2.5 mg/L,5 mg/L,10mg/L,20 mg/L,and 40 mg/L）in vitro.The results showed that the SKOV3 cells to Bevac The monoclonal antibody was dose-dependent with an IC50 value of（8.89±0.27）mg/L.The combination of bevacizumab and 3 mg/L cisplatin significantly inhibited the proliferation of ovarian cancer cells.3.The effects of bevacizumab and cisplatin alone and in combination on the invasion of SKOV3.Transwell plots showed that the number of invasive cells in the bevacizumab-and cisplatin-alone drug-treated groups was lower than that in the control group,and the number of invasive cells in the combined-drug-treated group was significantly reduced,and the results were shown by a one-way analysis of variance.The difference between the drug treatment group and the drug treatment group was statistically significant（P<0.001）4.Effects of bevacizumab and cisplatin alone and in combination on the apoptosis of ovarian cancer cells.The apoptosis rate was（6.8±0.8）%in bevacizumab-treated group,（13.1±2.1）%in cisplatin alone group,and（38.0±2.2）%in the combination therapy group.One-way analysis of variance showed that the rate of cell apoptosis was significantly greater in the combination drug treatment group than in the drug treatment group alone,and the difference was statistically significant.5.The effects of bevacizumab and cisplatin alone and in combination on the expression of key proteins,MMP-9 protein and Bad protein in the downstream signaling pathway of VEGF.Cisplatin alone had no effect on the phosphorylation level of the key protein AKT in the downstream signaling pathway of VEGF.The use of bevacizumab alone attenuated the expression of AKT phosphorylation,but the combination of the two could significantly reduce the expression of phosphorylated AKT.And the expression of pro-apoptotic protein Bad was significantly increased in its downstream signaling pathway.The use of bevacizumab and cisplatin alone had little effect on the expression of MMP-9 protein,and the combination of the two could significantly reduce the expression of MMP-9 protein.Conclusions1.In ovary cancer tissues,VEGF and MMP-9 proteins present a common overexpression state,which plays a promoting role in the development of ovarian cancer.2.Bevacizumab combined with cisplatin inhibited SKOV3 cell proliferation,attenuated invasion,and induced apoptosis.3.Bevacizumab combined with cisplatin inhibits the conduction of VEGF downstream signaling pathways,thereby exerting a synergistic anti-tumor effect,providing a theoretical basis for the clinical treatment of rub.