| Ovarian cancer(OC)has become a serious threat to women’s health,with an increasing incidence.Because of its insidious onset,two-thirds of ovarian cancer patients have reached the middle and advanced stages at the time of detection,often accompanied by distant metastasis,with a recurrence rate as high as 70%and a very poor prognosis.There is an urgent need to find an ideal treatment plan to improve the status of patients with ovarian cancer.Traditional tumor cell reduction combined with platinum-based chemotherapy has always been used as the standard treatment for ovarian cancer in Western medicine.But the subsequent surgical trauma,drug side effects,and drug resistance to platinum-based chemotherapy drugs cannot be ignored.In recent years,the research of new targeted drugs has become a new hotspot in the field of ovarian cancer.Poly(ADP-ribose)polymerase(PARP)inhibitors are typical representatives of ovarian cancer targeted drugs.PARP is involved in a variety of biological processes such as cell cycle regulation,cell differentiation and cell replication,among which the most important is the repair of DNA single strand damage.The main role of PARP inhibitors is to prevent the self-damage repair of single-stranded DNA in tumor cells,resulting in DNA double-strand break at the replication fork.If the tumor cell susceptibility gene BRCA is absent at this time,the tumor cells can neither carry out homologous recombination repair nor repair single chain through PARP,which will result in "synthetic lethality" effect on tumor cells,resulting in the death of tumor cells.Olaparib is the most widely used and earliest PARP inhibitor.A large number of clinical prospective and retrospective datas have confirmed that olaparib can significantly prolong the progression-free survival of ovarian cancer patients,especially those with BRCA 1/2 mutations.Olaparib has been approved by the European Commission for platinum-sensitive recurrent high-grade ovarian cancer patients with BRCA1/2 mutations.However,only 5%-10%of ovarian cancer patients have congenital BRCA1/2 mutations,and how to expand the use of PARP inhibitors has become a new research field.Some clinical trials have found that PARP inhibitors can achieve synergistic effects when combined with chemotherapy drugs such as alkylating agents in the treatment of solid tumors,which directly damage the DNA strand by alkylating agents.Studies have shown that the combination of PARP inhibitors and DNA damage repair inhibitors can increase the sensitivity of tumor cells to PARP inhibitors,improve the resistance of PARP inhibitors,and synergistically inhibit the growth of tumor cells.It has also been found that vascular endothelial growth factor inhibitors can induce the down-regulation of homologous recombination repair-related proteins BRCA1/2 and RAD51,and enhance the sensitivity of PARP inhibitors when used in combination with PARP inhibitors,and this effect is independent of BRCA status.These provide a basis for the feasibility of PARP inhibitor combination therapy.With the development of traditional Chinese medicine(TCM),the purification technology of TCM has been continuously improved,and the excavation and clinical application of classical prescriptions of traditional Chinese medicine have made continuous progress in the treatment of diseases.After three years of COVID-19 epidemic,TCM has received unprecedented attention.We can clearly see the broad scope and unexplored potential value of TCM.At present,western medicine treatment is still the main way to treat cancer.However,we can take advantage of TCM’s advantages of multiple targets,high efficiency,low toxicity and low cost to assist Western medicine in treatment.Although there is no term "ovarian cancer" in TCM,however,ovarian cancer could be classified as "lump","lump","lump of intestine",etc,based on its clinical manifestations.Many TCM experts believe that the pathogenesis of ovarian cancer is "deficiency of vital Qi" and "invasion of positive deficiency and pathogenic factors",and the treatment should be based on"strengthening the body and eliminating pathogenic factors",and "strengthening the body and strengthening the root" or "supplementing Qi and clearing poison".So tonifying Qi drugs are widely used in traditional Chinese medicine prescriptions for treating ovarian cancer,especially Astragalus membranaceus(AM).At present,there are few reports on the molecular mechanism of AM in the treatment of ovarian cancer,including the main active ingredients that exert anti-cancer effects.Therefore,AM is the first choice for our study.This study is divided into three parts:At first,we explored the main active ingredients of AM,predicted the key anti-ovarian cancer targets of AM,further optimized related targets to explore the signal pathways through protein interaction analysis,enrichment pathway analysis and molecular interconnection verification,based on network pharmacological methods.Finally,quercetin was selected as the main research object for subsequent experiments.Second,the inhibitory effect of quercetin alone or in combination with PARP inhibitor olaparib on ovarian cancer A2780 and SKOV3 cells and its potential mechanism were investigated in vitro.Finally,the effect of quercetin alone or in combination with olaparib on the growth of ovarian cancer in vivo and the expression of proteins involved in homologous recombination repair pathway were further verified by subcutaneous xenograft in nude mice.Part Ⅰ To explore the mechanism of Astragalus membranaceus in ovarian cancer based on network pharmacology and molecular dockingObjective:The active ingredients,anti-ovarian cancer targets,signaling pathways and pharmacological mechanisms contained in AM were studied,through the network pharmacology of TCM.Methods:1.The main active ingredients of AM were obtained by TCMSP,and the conditions were OB≥30%,DL≥0.18 and HL≥4 hours.2.The targets of the main active components of AM were obtained from Uniprot database.3.Gene Cards,DisGeNET and Open Targets were used to screen the relevant targets of ovarian cancer.4.AM-OC intersection target was obtained by Venny2.1.0 online Venn diagram.5.The AM-composition-targets-OC relationship network diagram was constructed by Cytoscape 3.8.0 software,and core components were screened.6.The PPI network diagram of intersection targets was drawn using STRING 11.5 database and loaded on Cytoscape 3.8.0 software to construct the network.And CytoHubba plug-ins and MCODE plug-ins were used to screen key targets.7.David database was used for GO biological function and KEGG pathway enrichment analysis to screen core pathways..8.AutoDock Vina 1.1.2 was used for docking,and PyMOL2.3.0 was used to analyze the interaction mode of docking results,to verify the affinity between the core targets and core active component.Results:1.The active constituents and targets of AM and related targets of ovarian cancer were obtained,and "AM-OC" intersection targets were screened.A total of 87 AM components were obtained from the TCMSP database,and 16 major active components of AM and 184 corresponding targets were screened according to the setting.GeneCards,DisGeNET and Open Targets databases were searched,and the top 149 targets in each database were selected.A total of 28 "AM-OC" intersection targets were obtained by drawing Wayne diagram.2.Quercetin is the core active component of AM in the treatment of ovarian cancer.By analyzing the "AM-components-intersection target-OC" relationship network,28 targets were associated with 14 components,and the main active ingredients were quercetin,kaempferol,formononetin,isorhamnetin,and calycosin,which were connected with 24,9,8,8,and 7 targets,respectively.Quercetin,as a core component,will be the subject of study in subsequent cell and animal experiments.3.The key targets of AM against ovarian cancer are P53,PTEN,VEGFA,MYC,AKT1 and CCND1.Through the PPI network visualization analysis of the "AM-OC"intersection targets,the key targets were screened according to the Degree value,suggesting that these targets may be the potential targets of AM against ovarian cancer.CytoHubba and MCODE plug-ins were used to verify the key targets again.4.The biological functions of AM against ovarian cancer are rich and there are many pathways,reflecting the characteristics of Astragalus membranaceus’s anti-ovarian cancer effect of "multi-component,multi-target and multi-pathway".The DAVID database was used to perform GO and KEGG enrichment analysis of the 28 common targets,and the top 10 GO biological function items and the top 20 KEGG pathway enrichment analysis items were selected.GO function included enzyme regulation,angiogenesis,oxidation,etc.KEGG pathway enrichment included a variety of cancer pathways,and the target-pathway network map was constructed.5.Quercetin has the strongest and most stable binding to MYC,and the results indirectly prove that quercetin can play a regulatory role in the core target.Molecular docking was performed between core targets(TP53,PTEN,VEGFA,MYC,AKT1,CCND1)and core component—quercetin.The highest absolute binding energy was MYC and quercetin,and the binding energy was-8.1 kal/mol.Conclusion:1.The results of network pharmacology analysis suggest that AM can exert anti-ovarian cancer effects through multiple targets and pathways,which provides a reference for further anti-cancer research of ovarian cancer.2.The results of network pharmacology analysis suggested that quercetin was the most important active component of AM against ovarian cancer,which provided an important basis for further in vitro and in vivo biological experiments.3.GO and KEGG enrichment analysis showed that the potential pathways of AM against ovarian cancer were diverse and complex.Among them,cancer pathways involve multiple targets.Part Ⅱ To investigate the effects of quercetin combined with olaparib on the proliferation,migration and apoptosis of ovarian cancer cells A2780 and SKOV3 and its potential mechanismObjective:Quercetin,the core component of Astragalus membranaceus against ovarian cancer obtained by network pharmacology and molecular docking in the first part,was used to inhibit the growth of ovarian cancer A2780 and SKOV3 cells.Quercetin combined with olaparib was used to explore the effect and mechanism of the two drugs combined against ovarian cancer.Methods:1.Culture of ovarian cancer cell lines A2780 and SKOV3 In vitro.2.CCK8 assay was used to detect the effects of quercetin and olaparib on the viability of SKOV3 and A2780 cells.3.Annexin V-FITC/PI double staining was used to detect the apoptosis of SKOV3 and A2780 cells treated with quercetin or olaparib alone or in combination.4.Scratch test and transwell migration test were used to detect the effects of quercetin,olaparib and their combination on the migration ability of SKOV3 and A2780 cells.5.Colony formation assay was used to detect the effects of quercetin and olaparib alone or in combination on the colony formation ability of SKOV3 and A2780 cells.6.Western blotting was used to detect the expression of DNA repair related proteins(ATM,p-ATM,BRCA1,RAD51,PARP1,OGG1)in A2780 and SKOV3 cells after treatment by quercetin,olaparib and their combination,in order to analyze the effect of quercetin and their combination on DNA damage repair and explore the mechanism of action.Results:1.Quercetin,olaparib,and their combination inhibited the proliferation of ovarian cancer A2780 and SKOV3 cells.The inhibitory activities of quercetin and olaparib on A2780 and SKOV3 cells were in dose-and time-dependent.The inhibition rate increased with the increase of concentration and time.The inhibitory effect of quercetin combined with olaparib on ovarian cancer cells was significantly higher than that of quercetin or olaparib alone.2.Quercetin,olaparib and their combination promoted apoptosis of ovarian cancer A2780 and SKOV3 cells.Flow cytometry showed that the apoptosis of quercetin and olaparib combination group was mainly in late apoptosis.In both ovarian cancer cells,total apoptosis rate in combination group was significantly higher than that of single drug group.3.Quercetin,olaparib and their combination inhibited the migration of ovarian cancer A2780 and SKOV3 cells.The cell scratch healing and transwell experiments showed that,compared with the control group and the single drug group,quercetin combined with olaparib significantly inhibited the migration ability of A2780 and SKOV3 cells.The combined drug group had the slowest scratch healing,the largest scratch area,and the least number of migrated cells.4.Quercetin,olaparib and their combination inhibited the clonogenicity of ovarian cancer A2780 and SKOV3 cell Clone.The results of colony formation assay showed that quercetin combined with olaparib significantly inhibited the colony formation of A2780 and SKOV3 cells.5.Quercetin down-regulates PARP1 expression in ovarian cancer A2780 and SKOV3 cells.Western blot results showed that quercetin treatment decreased the expression of PARP1 protein in A2780 and SKOV3 cells compared with olaparib treatment,while olaparib treatment increased the expression of PARP1 protein.In terms of affecting PARP1 gene expression,the two have opposite effects.6.Quercetin and their combination inhibited the expression of homologous recombination repair-related proteins(ATM,p-ATM,BRCA1,RAD51)in ovarian cancer A2780 and SKOV3 cells.Western blot results showed that quercetin inhibited the expression of homologous recombination repair-related proteins(ATM,p-ATM,BRCA1,RAD51)and hindered the process of homologous recombination repair.Quercetin combined with olaparib exerted synthetic lethal effect.7.Quercetin up-regulates the expression of OGG1 gene in A2780 cells and SKOV3 cells.Western blot results showed that OGG1 expression in A2780 cells was up-regulated in Que group compared with the other three groups,and the expression differences were significant(P<0.05 or P<0.01).In SKOV3 cells,the expression of OGG1 was up-regulated in Que group,but there was no significant difference among all groups(P>0.05).Conclusion:1.Quercetin or olaparib can effectively inhibit the proliferation of ovarian cancer A2780 and SKOV3 cells in a time-and concentration-dependent manner.2.Quercetin combined with olaparib can enhance the effects of single drug on A2780 and SKOV3 cell proliferation,induction of cell apoptosis,inhibition of cell migration and colony formation.3.Quercetin can inhibit the expression of PARP1 protein and repair the homologous recombination of ovarian cancer cells.In combination with olaparib,quercetin has a synthetic lethal effect on ovarian cancer cells.Part Ⅲ Effects of quercetin combined with olaparib on the growth of ovarian cancer xenografts in nude mice and its mechanismObjective:Quercetin has been shown to play an important role as a tumor suppressor in ovarian cancer by network pharmacology and cell experiments.In order to verify the effect of quercetin on the growth of ovarian cancer in vivo,nude mice were used to determine the effect and mechanism of quercetin and quercetin combined with olaparib on the growth of ovarian cancer in vivo.Methods:1.Nude mice were inoculated with A2780 and SKOV3 cells for tumor formation experiments.The growth of subcutaneous transplanted tumor was observed dynamically,and the volume of transplanted tumor and the body weight of nude mice were detected.At the end of the treatment,the nude mice were sacrificed,the tumor weight was recorded,and the tumor tissue was made into sections.2.Immunohistochemical staining was used to detect the expression and localization of ATM,p-ATM,BRCA1,RAD51,PARP1 and OGG1 proteins in the xenograft tissues of nude mice after treatment with quercetin or olaparib alone or in combinationResults:1.Compared with the control group,there was no significant difference in the growth status of the nude mice with ovarian cancer transplant tumor in each medication group.During the animal experiment,there was no significant difference in the weight increase,mental state and diet status of the nude mice with ovarian cancer transplant tumor in each medication group.2.The combination of quercetin and olaparib has the strongest anti-tumor effect.In A2780 nude mice,the tumor inhibition rate of olaparib group was 52.5%,that of quercetin group was 35%,and that of olaparib combined with quercetin group was 53.75%.The inhibition rates of olaparib,quercetin and Ola+Que groups were 27.27%,36.36%and 45.45%,respectively.Compared with the control group,the quercetin combined with olaparib group had the smallest tumor weight(P<0.01).3.The combination of quercetin and olaparib can significantly inhibit the expression of ATM,p-ATM,BRCA1 and RAD51 in ovarian cancer xenograft tissues.Immunohistochemical staining showed that ATM,p-ATM,BRCA1 and RAD51 proteins were mainly expressed in the nucleus of ovarian cancer xenografts,and some were also expressed in the cytoplasm.Compared with the control group,the combination of quercetin and olaparib significantly reduced the expression of the above proteins,especially ATM,p-ATM and RAD51 proteins,showing a small proportion of nuclear staining.4.Quercetin down-regulates the expression of PARP1 protein in ovarian transplanted tumor tissues.Immunohistochemical staining showed that PARP1 protein was mainly expressed in the nucleus of ovarian cancer xenografts.Compared with olaparib,Quercetin treatment reduced the expression of PARP1 in ovarian cancer xenografts,while olaparib treatment increased the expression of PARP1.5.Quercetin up-regulates OGG1 gene expression in ovarian xenograft tissues.Immunohistochemical staining showed that Quercetin up-regulated the expression of OGG1 protein in ovarian cancer xenograft tissues compared with the control group.Conclusion:1.Animal experiments showed that quercetin combined with oraparil had stronger inhibitory effect on the growth of ovarian cancer cells A2780 and SKOV3 subcutaneous grafts in nude mice than single drug..2.Quercetin combined with olaparib can inhibit the expression of PARP1,ATM,p-ATM,BRCA1 and RAD51 in transplanted tumor tissue,and play a synthetic lethal role,which is independent of the homologous recombination state of tumor itself. |
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