| Ovarian cancer is the malignant tumor with the highest fatality rate among female reproductive tract tumors,and its incidence ranks the third among gynecological malignant tumors.Epithelial ovarian cancer(EOC)is the most common pathological type of ovarian cancer,its onset is hidden,most patients are diagnosed in late stage;and prone to invasion and metastasis,more than 80%of EOC patients have extensive metastasis at the time of diagnosis;at the same time,surgery is easy to relapse,chemotherapy is easy to be resistant,and about 70%of patients relapse within 3 years of treatment.Therefore,it is of great significance to explore the pathogenesis and metastasis of EOC and to find new targets and treatment methods to improve the survival rate of EOC patients.Metastasis and chemotherapy resistance are the main obstacles to the treatment of ovarian cancer.Epithelial-mesenchymal transformation(EMT)is a biological process in which epithelial cells lose their cell polarity and adhesion and obtain the characteristics of migration,invasion and metastasis of mesenchymal cells.EMT is regulated by complex molecular pathways and involves a variety of cellular signal pathways,among which TGF-β signal transduction pathway mediated by intracellular signal transduction protein Smad is a classical pathway.EMT contributes to tumor metastasis,chemotherapy resistance and immunosuppression.Therefore,targeting EMT is an attractive method for cancer treatment,and drugs that reverse epithelial-mesenchymal transformation are of great significance to improve cancer treatment.Adenosine diphosphate ribosylation factor guanylate kinase 1(ASAP1)is a kind of GTP enzyme activating protein containing SH3,ANK repeats and PH1 domain,which participates in the regulation of cytoskeleton rearrangement and cell membrane movement,and plays an important role in the migration and diffusion of tumor cells.Studies have shown that ASAP1 is up-regulated and promotes EMT in a variety of cancers,and is associated with poor survival and prognosis of patients with thyroid cancer,breast cancer,colorectal cancer and prostate cancer.In ovarian cancer,the high expression of ASAP1 promotes tumor proliferation,invasion and metastasis,indicating that ASAP1 is a potential biomarker for diagnosis and prognosis of patients with ovarian cancer.HL142 is a kind of UCS15A analogue synthesized by ourselves.UCS15A is an antibiotic extracted from Streptomyces.As an inhibitor of SH3/proline binding,UCS15A has been shown to destroy the intermolecular interaction mediated by SH3 domain in eukaryotic cells.ASAP1 has been shown to induce tumor metastasis and chemotherapy resistance by interacting with the PH or PH3 domains of interacting proteins.Therefore,we speculate that HL142 can inhibit the proliferation,invasion and metastasis of ovarian cancer cells by destroying the SH3 and PH domains of the oncoprotein ASAP1 interacting with other carcinogenic proteins.HL142 is expected to become a new and effective drug for the treatment of ovarian cancer.Objective(1)To explore the significance of ASAP1 in the diagnosis and treatment of ovarian cancer by studying the role and mechanism of ASAP1 in ovarian cancer.(2)To clarify the effect of HL142 on ASAP1,analyze the effect and mechanism of HL142 on ovarian cancer cells,and verify the therapeutic effect and mechanism of HL142 on ovarian cancer through animal experiments,and to explore the application prospect of HL142 as a new type of ovarian cancer therapy drug.Material and methods1 Object of studyCell lines:Ovarian cancer cell lines OVCAR3 and OVCAR8 were purchased from National Cancer Institute;HEK293 FT cell was obtained from ATCC.Animals:Cg-Prkdcscid Il2rgtm1Wjl/Sz J(NSG)female immunodeficient mice were purchased from Jackson Laboratory in the United States.2 Methods(1)Cell culture:Both OVCAR3 and OVCAR8 cells were cultured in RPMI 1640 medium supplemented with 10%FBS(Hyclone,Logan,UT),100μg/ml streptomycin and 100U/ml penicillin(Invitrogen,Carlsbad,CA);HEK293FT cells were cultured in DMEM(Dulbecco’s Modified Eagle Medium)containing 1%glutamine,10%FBS,100μg/ml streptomycin and 100U/ml penicillin.(2)Construction of gene knockout cell lines:The CRISPR/Cas9 system was used to construct the ASAP1 gene knockout vector,which was packaged and purified by lentiviral vector system and transfected into ovarian cancer cells.Ovarian cancer OVCAR3 and OVCAR8 cell lines with gene knockout of ASAP1 were constructed and introduced into the empty vector virus as the control group.(3)Western blotting:Protein was extracted from ovarian cancer cells and tumor tissues of mice in ASAP1 knockout group and control group,and the expression of ASAP1 and EMT-related proteins was detected.The same method was used to detect the expression of AS API,FAK,EMT-related proteins,TGF-β pathway-related proteins,FAK pathway-related proteins and apoptosis proteins in ovarian cancer cells or tumor tissues treated with HL142 and vehicle.(4)MTT and colony formation assay:The growth and proliferation rate of cells in ASAP1 knockout group and control group were detected,and the proliferation ability of ovarian cancer cells treated with HL142 and vehicle was detected by the same method.(5)Cell migration and invasion test:The migration and invasion ability of ovarian cancer cells in ASAP1 knockout group,control group,HL142 group and vehicle group were detected.(6)Immunoprecipitation:the interaction between AS AP1 and FAK was detected.(7)Animal experiment:The orthotopic transplantation tumor model of ovarian cancer in nude mice was established by 8-week-old female immunodeficient mice.The occurrence,development and metastasis of ovarian cancer were monitored by Xenogen imaging system.The tumors of ovarian cancer in situ and metastatic tumors in organs were collected,and the size and weight of tumors were measured to observe the effects of ASAP1 and HL142 on the growth and metastasis.(8)Immunofluorescence staining:Tumor tissues in mouse ovaries and metastatic organs were collected and paraffin sections were made.ASAP1,cytokeratin-7 and vimentin were stained with immunofluorescence and photographed under microscope to detect the expression of ASAP 1,cytokeratin-7 and vimentin in tumor tissues.(9)HE staining:The ovaries and organs of mice were collected and paraffin sections were made.The sections were stained with HE and photographed under microscope to observe the metastasis of tumor in the organs of mice.3.Statistical analysesThe experimental data were analyzed by GraphPad Prism 7 statistical software.The quantitative data were expressed by mean ±standard deviation(X±S).The differences between the two groups were compared by independent sample t test.Differences between multiple groups were compared by single factor analysis of variance(one-way ANOVA),followed by LSD-t pairwise comparison.α=0.05 was used as the test level,P<0.05 was statistically significant.Results(1)Western blotting showed that compared with the control group,the expression of EMT-related interstitial markers N-cadherin,β-catenin,vimentin and snail2 decreased,while the expression of epithelial markers E-cadherin and cytokeratin-7 increased in ASAP1 knockout group.Compared with vehicle group,the same experimental results were obtained in HL142 group,and the expression of ASAP1 and FAK was significantly decreased in HL142 group.(2)The results of cell function experiment showed that the ability of cell proliferation,colony formation,migration and invasion in ASAP1 knockout group were significantly lower than those in control group.Compared with vehicle group,HL142 group got the same experimental results.(3)OVCAR3 and OVCAR8 cells were treated with HL142 and vehicle for 6 hours,then the cells were treated with 6 ng/ml TGF-β at different time points and the proteins were extracted for western blotting.The results showed that the expression of phospho-SMAD2 and total-SMAD2 decreased in HL142 group,and the decrease of phospho-SMAD2 was more significant.(4)OVCAR3 and OVCAR8 cells were treated with vehicle,HL142,paclitaxel and HL142 combined with paclitaxel,respectively.The expression levels of apoptosis-related proteins cleaved-PARP and cleaved-caspase3 were detected by Western blotting.The results showed that HL142 significantly induced apoptosis in the two cell lines and made the cells sensitive to paclitaxel treatment.(5)OVCAR3 and OVCAR8 cells were treated with HL142 and vehicle for 6 hours,then 10 μM RGD was used to activate FAK pathway at different time points,then the protein was extracted for Western blotting.The results showed that HL142 could significantly weaken the FAK pathway activated by RGD.(6)Xenogen imaging system monitoring tumor imaging results showed that the volume and weight of tumors in the ASAP1 knockout group were smaller than those in the control group,and there was no distant organ metastasis.Compared with the vehicle group,the HL142 group obtained the same experimental results.(7)Western blotting was used to detect the expression of related proteins in mouse tumor tissues in situ,and the results were consistent with the data obtained by cell line experiments.(8)HE staining results of mouse tumor sections showed that liver,spleen and kidney metastasis occurred in the control group and vehicle group,while no distant organ metastasis was found in the ASAP1 knockout group and HL142 group.(9)The immunofluorescence results of tumor sections of mice showed that the expression of ASAP1 and vimentin decreased and the expression of cytokeratin-7 increased in HL142 group and ASAP1 knockout group.Conclusions(1)ASAP1 enhances the proliferation,invasion and migration of ovarian cancer cells by promoting EMT.ASAP1 is a potential biomarker for diagnosis and prognosis of patients with ovarian cancer.(2)UCS15A analogue HL142 inhibits the growth and metastasis of ovarian tumor by inhibiting the expression of ASAP1 and FAK,reversing EMT,weakening TGF-β and FAK pathway.(3)HL142 induces apoptosis of ovarian cancer cells and enhances the efficacy of paclitaxel,which is expected to become a new and effective drug for the treatment of ovarian cancer. |
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