Experimental Study Of Radionuclide Imaging And Inhibiting Cell Activity By Targeting VEGFR-2in Ovarian Cancer

ObjectiveOvarian cancer is one of the most common gynecologic cancers. Its early clinical symptoms is not obvious, and early detection lack of reliable method,75%of ovarian cancers were diagnosed in late stage and had abdominal metastasis, which had lost the opportunity of surgical ablation. The five-year survival rate is only20%-30%. Ovarian cancer is the leading cause of death from gynecological malignancy. But the five-year survival rate of ovarian cancer patients diagnosed in early stage is up to90%. The standard therapy is cytoreductive surgery followed by cisplatin-based chemotherapy. But the relapse rate and resistance rate is high, survival has not been effectively improved. Therefore, to seek a specific target for early diagnosis and effective treatment of ovarian cancer has become a hot research. Studies have revealed that the vascular endothelial growth factor receptor-2/vascular endothelial growth factor (VEGFR-2/VEGF) signaling pathway involves in cell migration, proliferation and survival of nascent endotheliocyte around tumor, and plays a central role in tumor vasculature of ovarian cancer. VEGF has many forms of protein, and the most significant proangiogenic is VEGF165. VEGFR-2is one of the main receptors that mediated VEGF165promote angiogenesis, and it plays a central role in signal transduction and vascular endothelium generation. Moreover, VEGFR-2is highly expressed in ovarian cancer tumor tissue. This study plans to establish ovarian tumor-bearing models in nude mouse, to investigate whether VEGFR-2could be used as a diagnostic biomarker for ovarian cancer using autoradiography imaging method with131I-VEGFi65.(S, R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) is a competitive inhibitor of tautomerase activity as well as an specific inhibitor of MIF. It can selectively combined with the active site of MIF tautomeric isomerase, and significantly inhibit MIF activity. But it has not been reported that whether ISO-1can regulate the VEGFR-2level through effect the activities of MIF in ovarian cancer. This study investigate the effect of different concentrations of ISO-1on the proliferation、invasion and VEGFR-2level of ovarian cancer cells, and provide experimental evidence for the VEGFR-2-targeted treatment of ovarian cancer.MethodsVEGF165was radioiodinated with Na131I by Iodogen method,131I-VEGF165were separated from free iodine using Sephadex G-25columns. The radiochemical purity of radioiodinated VEGF165was got by paper chromatography. At the same time, the uptakes of I31I-VEGF165by ovarian cancer cells were analyzed. The ovarian tumor-bearing models were established, and when the tumor volume reached1cm3(6weeks after inoculation), the tumor-bearing nude mice were used for autoradiography imaging studies. The tumor targeting and body distribution of ovarian tumor-bearing models was analyzed after injection of131I-VEGF165for3h、6h and8h. Using RT-PCR, Western blot and immunohistochemical assay of tumor tissue to detect VEGFR-2expression.The human ovarian cancer cell lines SKOV3and A2780were treated with series concentrations of ISO-1. MTT assay was used to measure the cell proliferation. The tautomerase activity of MIF was evaluated by using L-dopachromemethyl ester. Microporous migration assay w as performed to determine the effect of ISO-1on the invasion of SKOV3and A2780cells. Fluorescence was used to test apoptosis. The expression of mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of protein was detected by cell immunochemistry.ResultsThe labeled rate of131I-VEGF,65was90.47%, radiochemical purity was94.17%, and the immune activity of markers was well. The uptake of131I-VEGF165by human ovarian cancer cells SKOV3was much higher than that of control group (Na131I) at0.5、1、2、4and24h (P<0.05).The target-to-non-target (T/NT) ratios were2.72±0.19(3h),4.85±0.81(6h), and3.31±0.57(8h).RT-PCR and Western Blot revealed thatVEGF-165, VEGFR-2were detected in the ovarian carcinoma tissues and SKOV3cells not in the controls. Wholebody autoradiography showed that uptake in well-perfused organs at3h and clear tumor localization from6h onward, these findings were in accordance with the high T/NT ratio.ISO-1inhibited the proliferation and the MIF tautomerase activities of SKOV3and A2780cells obviously (P<0.05).After treated with50μmol/L ISO-1for24h, the ability to penetrate the polycarbonate film is significantly reduced (P<0.05), apoptosis is obviously. meanwhile, the mRNA and protein level of VEGFR-2and VEGF is also reduced (P<0.05)ConclusionsVEGF165was easily to be iodinated with131I by Iodogen method, and131I-VEGF165was stable performance.131I-VEGF165can targeting gathered at the tumor site, and the autoradiography imagings were clearly. Based on our experience, the tumor uptake of131I-VEGF165measured by wholebody autoradiography reflects tumor VEGFR-2expression level in vivo. ISO-1can down-regulate the expression of VEGFR-2and VEGF, thus inhibit the proliferation and invasion of ovarian cancer cells. It provides the experimental evidence for the targeted therapy of VEGFR-2for ovarian cancer.