| Background:Ovarian cancer is a common gynecological malignant tumor which has top mortality rates.Among of ovarian cancer,epithelia ovarian cancer is the main type of all malignant ovarian tumors.Surgery and chemotherapy are very effective in reducing tumor burden,however,relapses occur frequently and there is a lack of credible second-line options.Therefore,new treatment with antibody has been brought up according to ovarian cancer pathological features,a variety of antibodies have get in clinical research.Preliminary results of these treatment show efficacy in some patients,however,there are antibody resistance appear which induce poor efficacy and Tumor recurrence.Therefore,investigate the molecular mechanism of antibody resistance,looking for effective intervention strategy has great significance.Previously,we established a trastuzumab-resistant ovarian cancer cell line,named as SKOV3-T,which was developed through continuously culturing SKOV3 cells in the presence of trastuzumab.VEGF is a well-known angiogenesis factor involved in many physiological and pathological processes.Its significance has been implicated in promoting tumor growth and metastasis via angiogenesis.Objective:In acquired trastuzumab-resistant ovarian cancer model investigate drug resistance mechanism of drug resistance of tumor,aims to find out the ways to overcome resistance.Method:First we compare the capacity of tumorigenesis and angiogenesis SKOV3 with SKOV3-T by using Western Blot,cell proliferation,and tumorigenesis.We found that VEGF was up-regulated in SKOV3-T Cells by using cell growth/proliferation,RT-PCR,Transwell assay and ELISA.Furthermore,overexpression of VEGF in SKOV3-T is connected with the migration and angiogenesis of SKOV3-T.Then,we explore the mechanism of malignant degree increased by using Western Blot,Nuclear plasma separation experiment and confocal microscopy.Then transfering SP1 shRNA plasmid into SKOV3-T to knockdown SP1,we explore the SP1 regulation of the expression of VEGF and verify SP1 potential role in the HER2 antibody resistance.Eventually,we screened two SP1-knockdown clones,3D7 and 1F10,and explored the influence of SP1-knockdown inhibited the tumor growth and angiogenesis of SKOV3-T in vivo by using ELISA and tumorigenesis.Results:SKOV3-T xenografts were obviously bigger than SKOV3,while the shape was irregular to suggest possible enhanced angiogenesis;what'more,the microvessel density of SKOV3-T xenograft was higher than that of SKOV3 tumor specimens;It suggested Trastuzumab-resistant SKOV3-T cells possesses enhanced tumorigenesis and angiogenesis.HUVECs proliferation could be enhanced by SKOV3-T cell supernatant and VEGF secretion of SKOV3-T cells was higher than SKOV3;Meanwhile' Anti-VEGF antibody(Avastin)could inhibit the enhanced migration activity of SKOV3-T on a dose dependent manner;So we concluded overexpression of VEGF in SKOV3-T accelerated the migration of SKOV3-T.Up-regulated SP1 in the nuclear might be the key transcriptional factor to induce the over-expression of VEGF in SKOV3-T Cells,Suggesting SP1 knockdown inhibited VEGF-related migration in SKOV3-T cells.Finally,SP1-knockdown inhibited the tumor growth and angiogenesis of SKOV3-T in vivo.Conclusion:After ovarian targeted therapy antibody resistance in the process,the expression of tumor related biomarkers SP1 was induced to rise,show the antibody resistant cell SKOV3-T cells,the tumor angiogenesis,migration ability and tumorigenesis in vivo were significantly stronger than the original generation of ovarian cancer cell SKOV3.VEGF secretion of SKOV3-T cells was higher than SKOV3,what'more,overexpression of VEGF in SKOV3-T accelerated the migration and tumor angiogenesis of SKOV3-T.SKOV3-T cell transfection SP1 shRNA knockdown SP1,the capacity of migration ability and tumorigenesis in vivo decreased,shows that SP1 was one of the important factors,suggesting SPlcan be a good target targeted therapy in the future,and provide a basis for the combination therapy of multiple targets. |
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