Study Of The Anti-Tumor Effect And Mechanisms Of Metformin Combined With MDM2 Inhibitor RG7388 Against Ovarian Cancer

BackgroundOvarian cancer is one of the common gynecologic malignancies,and it is the most mortality gynecological cancer,which seriously threatens the health of women.According to the latest statistics,in the United States,the number of confirmed ovarian cancer in 2020 is 21750,the death numble is 13940,and the 5-year survival rate of advanced diseases is about 30%.However,most of the patients with epithelial ovarian cancer were diagnosed at the late stage of the disease because of the occultation of the disease.At present,the initial treatment of the disease is still a comprehensive treatment scheme of tumor cell reduction,with platinum combined with taxol as the auxiliary treatment.Although radical surgery and high response rate to initial chemotherapy were achieved,up to 70%of patients with ovarian cancer would have relapse of disease,and the median progression free survival period was only 12-18 months.Moreover,with the emergence of platinum resistance and refractory diseases,the sensitivity to chemotherapy of platinum drugs decreased with the subsequent recurrence.In addition,chemotherapy,such as neurotoxicity,joint pain and fatigue,can have a negative effect on the quality of life of patients.Therefore,it is urgent to have new targeted drugs and treatment approaches to improve the clinical effect and tolerance of ovarian cancer patients and improve the quality of life of patients.PARP inhibitor is a target drug approved for women with recurrent epithelial ovarian cancer.Early clinical data show that PARP inhibitor is effective for patients with BRCA1/2 mutation and platinum sensitive ovarian cancer,which can significantly improve the survival rate of patients after operation.Considering that ovarian cancer is a heterogeneous disease with complex molecular and genetic changes,the targeted treatment involves various pathways,so single inhibitor is unlikely to achieve satisfactory treatment effect and is prone to drug resistance Therefore,more and more attention has been paid to the combination of different drugs.It has been confirmed that most solid tumors are characterized by "Warburg effect",that is,even well oxygenated cancer cells have high glucose consumption and high lactate production,which indicates that glycolysis is up regulated.In view of this abnormal phenomenon,in addition to traditional cytotoxic drugs,drugs that affect the metabolism of tumor provide a new treatment idea for cancer treatment.More and more attention has been paid to the potential way to achieve a major breakthrough in cancer treatment and/or adjuvant treatment by targeting abnormal metabolism of tumor cells without causing toxic damage to normal cells.Glycolysis inhibitors may provide additional attack lines in combined therapy and play an important role in chemoresistance of tumor cells.Therefore,glycolysis inhibitors may make tumor cells sensitive and improve the results of conventional chemotherapy.Meanwhile,the combination of chemotherapeutic drugs and glycolysis inhibitors has been proved to be a promising strategy to overcome cancer resistance.Metformin(MET)is a recognized effective drug for the treatment of type 2 diabetes mellitus(T2DM).In view of the potential importance of Warburg effect in cancer metabolism,a glucose rich environment can provide favorable conditions for aerobic glycolysis.Therefore,MET therapy can reduce the hyperglycemia and related growth advantages of susceptible tumors.Epidemiological studies have identified the link between the use of met and the beneficial effects of cancer prevention and treatment,which makes people more and more interested in the potential antitumor effects of MET.In recent years,more and more research evidence shows that MET plays an important role in the adjuvant treatment of tumor,and can improve the sensitivity of traditional radiotherapy and chemotherapy and immunotherapy.But the anti-tumor mechanism of MET is not very clearP53 is one of the most important and best proteins in cancer.More and more evidence shows that members of p53 and its family play an important role in metabolism regulation.The activation of p53 can transcriptively inhibit the expression of GLUT1 or GLUT4,thus inhibiting glucose uptake.In addition,p53 also induces the expression of TIGAR(TP53 induces glycolysis and apoptosis regulators),witch is a transcription factor involved in the inhibition of glycolysis and pentose phosphate pathway.In contrast to wild type p53,mutant p53 promotes aerobic glycolysis by increasing GLUT1 membrane binding.Therefore,MET and p53 family proteins play an important role in regulating cancer metabolism,indicating that the function of p53 family protein has an internal relationship with the anti-cancer activity of MET.Since the discovery of the potential of p53 and its widespread inactivation in various cancers,many methods have been put forward to reactivate p53,and the development of small molecule MDM2 antagonists has become one of the main strategies in this field.RG7388(idasanutlin)is the second generation MDM2 antagonist,which has good efficacy,selectivity and oral bioavailability.It is suitable for clinical development to inhibit the interaction between MDM2 and p53 and activate p53 pathway.Preclinical studies confirmed that RG7388 as a single drug can lead to the increase of p53 gene activation,cell cycle arrest and apoptosis.In addition,RG7388 and cisplatin can play a synergistic role in the treatment of ovarian cancer.The current literature shows that MET can inhibit tumor occurrence through different signaling pathways in different cancer types,and it can regulate the expression and activity of p53 family protein in the process of tumor inhibition.For example,MET can inhibit the invasion and metastasis of melanoma by activating AMPK(monophosphate dependent protein kinase),which can induce phosphorylation and activation of p53.Met can also inhibit the expression of MDMX,reduce the interaction of MDMX-p53,and lead to the activation of p53 and cell death in lymphoma cells.The combination of MET and 2-deoxyglucose can induce apoptosis of prostate cancer cells in a p53 dependent manner.Some studies have shown that MET can inhibit the activity of cancer cells lacking p53 and play a cytotoxic role in the cells with abnormal p53 function.In addition,MET can make p53 deficient colorectal cancer cells sensitive to radiotherapy.These evidences show that p53 is involved in the antitumor effect of MET.Therefore,we assume that the combined action of MET and MDM2 antagonists may play a synergistic anti-tumor effect,but no relevant research reports have been reported in ovarian cancer.In this study,we studied the synergistic effect and the possible mechanism of MET combined with MDM2 antagonist RG7388 in different ovarian cancer cell lines.The research is divided into four parts:Objective:To explore the anti-cancer effects of MET combined with RG7388 against ovarian cancer cell lines A2780 and SKOV3.Methods:In this study,two different epithelial ovarian cancer cell lines A2780 and SKOV3 were cultured in vitro.The cells were treated with different concentrations of MET and RG7388,respectively.After 48 hours of treatment,cell growth was determined by CCK-8 assays,and the results of IC50 of MET and RG7388 on different cell lines were calculated respectively.The working concentrations of MET and RG7388 were determined in accordance with the IC50 values.The effects of MET,RG7388 alone or MET+RG7388 combined therapy on different ovarian cancer cells were observed for 48 hours.Cell proliferation was determined by CCK-8 assay and colony formation assay,and apoptosis was determined by Annexin V/PI double staining and Hoechst33258 staining.Results:1.Both MET and RG7388 alone and the combination of MET and RG7388 inhibited the growth of ovarian cancer cells,and the combination of the two drugs had synergistic inhibitory effect on the two cell lines.The average IC50 value of A2780 cells was 9.89 mM and that of SKOV3 cells was 41.7 mM.RG7388 repressed the growth of A2780 cells more effectively than that of SKOV3 cells.The average IC50 value of A2780 cells was 6.98?M(Fig.1B)and that of SKOV3 cells was>100?M.In accordance with the results of proliferation assays,we chose 5 mM Metformin and 3 ?M RG7388 as the drug concentrations for A2780 cells and chose 20 mM Metformin and 20 ?M RG7388 as the drug concentrations for SKOV3 cells in the follow-up experiments.CCK-8 results showed that the combination of MET and RG7388 led to greater growth suppression compared with either agent alone.2.Colony formation assays showed that the combination of MET and RG7388 inhibited colony formation of A2780 and SKOV3 cells more obviously compared with either agent alone.3.Both MET and RG7388 alone and the combination of MET and RG7388 promote the apoptosis of ovarian cancer cells.The effects of MET and RG7388 on apoptosis of ovarian cancer cells were assessed by flow cytometry and Hoechst staining.MET at 5 mM did not induce apoptosis of A2780 cells compared with the control.MET at 20 mM did not induce apoptosis of SKOV3 cells compared with the control.RG7388 at 3?M induced apoptosis of A2780 cells compared with the contro,and 20?M RG7388 did not induce SKOV3 cell death compared with the control.The combination of MET and RG7388 significantly increased apoptosis of both A2780(5 mM MET and 3?M RG7388)and SKOV3(20 mM MET and 20?M RG7388)cells compared with MET or RG7388 alone.Moreover,MET and RG7388 synergistically induced apoptosis of ovarian cancer cells as revealed by Hoechst staining of apoptotic bodies.The fluorescence intensity was weak and uniform in the control group,and bright blue apoptotic bodies were observed in experimental group.The number of apoptotic bodies in the cells treated with the combination was the highest and the fluorescence intensity was the highest.Conclusions:1.MET and RG7388 inhibited the proliferation and colony formation of ovarian cancer cells A2780 and SKOV3,and the combined effect of the two was significantly stronger than that of the drug alone2.MET and RG7388 alone can induce the apoptosis of ovarian cancer cells A2780,but not SKOV3.The combination of the two can obviously induce the apoptosis of A2780 and SKOV3 cellsObjective:To explore the possible mechanism of MET combined with RG7388 in inhibiting ovarian cancer cell lines.Methods:A2780 cells and SKOV3 cells were cultured in vitro.Different concentrations of MET and RG7388 were applied to the cells,respectively.After 48 hours of treatment,the expressions of p53 and the related proteins of PI3K/AKT/m-TOR pathway were detected by Western blot.Intracellular reactive oxygen species(ROS)levels were detected by flow cytometry.Results:1.MET and RG7388 can inhibit the expression of PI3K/AKT/m-TOR pathway-related proteins in ovarian cancer cells alone.In the combination group,phosphorylated PI3K,AKT,and mTOR were downregulated significantly compared with the control.2.MET and RG7388 increased the intracellular ROS level in A2780 cells.In SKOV3 cells,RG7388 increased the intracellular ROS level,whereas MET did not obviously increase the intracellular ROS level.The combination of MET and RG7388 increased the ROS level more obviously compared with either agent alone in both A2780 and SKOV3 cellsConclusions:1.Combination of MET and RG7388 can induce the accumulation of ROS in ovarian cancer cells and further induce the apoptosis.2.The combination of MET and RG7388 can inhibit the development of ovarian cancer by inhibiting the PI3K/AKT/mTOR signaling pathway.Objective:1.To establish a subcutaneous xenograft model of ovarian cancer in nude mice and investigate the anti-tumor effect of MET combined with RG7388 in vivo.2.To explore the mechanism of the anti-tumor effect in vivo of MET,RG7388 and their combination.Methods:A2780 cell suspension were implanted into the left axillae of each nude mouse.When palpable tumors had formed,the mice were randomly divided into four groups(n=5 per group):the control group,the MET group,the RG7388 group,and the MET+RG7388 group.Then,the mice were intraperitoneally injected everyday with MET(200 mg/kg/d)plus RG7388(10 mg/kg/d)or each alone for 7 days.PBS was used for the control.The xenografted tumor size was monitored every day with a sliding caliper and the volume was estimated using the following formula:volume=0.52×length×width2.After excision from the mice,the xenografted tumors were photographed and the PI3K/AKT/m-TOR pathway related proteins in the tissues were examined by western blotting.Results:1.Establishment of a subcutaneous xenograft model of ovarian cancer A2780 cells in nude mice:Cells were inoculated to the axillary skin of nude mice on the 4th day,nodules the size of small rice particles could be seen at the inoculation site,with clear boundaries and relatively tough texture,and the tumor formation rate of nude mice was 100%.On the 7th day of inoculation,the average diameter of the transplanted tumor was about 0.5cm.The transplanted tumor was randomly divided into groups and intraperitoneal injection of the drug was started.During the period of administration,the nude mice in each group were generally in good condition with normal diet and no obvious side effects or adverse reactions.2.The effect of combination of MET,RG7388 and MET+RG7388 on subcutaneous xenograft in nude mice with ovarian cancer:After 8 days of administration,the tumor was dissected and weighed.MET and RG7388 alone as well as their combination significantly decreased the xenografted tumor volume and weight.There was no significant difference in body weight and general conditions between the four groups,indicating that MET,RG7388 and the combination of the two drugs had no significant effect on the normal physiology of the nude mice.3.The mechanism of anti-tumor effect in nude mice.western blotting results indicated that MET and RG7388 alone as well as their combination downregulated the expression of phosphorylated PI3K,AKT,and mTOR compared with the control.Conclusions:1.MET and RG7388 can inhibit the growth of subcutaneous ovarian cancer xenografts in vivo,and their combination can inhibit the growth of the xenografts more obviously.2.MET and RG7388 inhibit the growth of xenografts by inhibiting the PI3K/AKT/m-TOR pathway in vivo3.The combination of MET and RG7388 did not affect the body weight or general physical condition of nude mice.Objective:Tandem mass spectrometry tagging(TMT)quantitative proteomics technology and bioinformatics analysis were used to study the specific mechanism of metformin on ovarian cancer cells,in order to explore a more effective combined regimen against ovarian cancer.Methods:A2780 cells and SKOV3 cells were cultured in vitro.After treatment with metformin at a certain concentration for 48 hours,the cells were lysed with SDT respectively and the protein samples were extracted.FASP method was used for peptide enzymolysis and TMT kit was used for labeling.After chromatographic separation,the samples were analyzed by MASS spectrometry with Q-EXactive mass spectrometer,and LC-MS/MS data were collected.Further application Complexheatmap R package for clustering analysis,protein with CELLO(http://cello.life.nctu.edu.tw/)to analyze subcellular localization method,with the Pfam database and InterProScan package domain of protein structure is analyzed.Blast2GO software and KAAS(KEGG Automatic Annotation Server)software were used to annotate the GO function and KEGG pathway of target proteins respectively.Fisher's Exact Test is used to carry out enrichment analysis of GO annotation or KEGG pathway annotation on target protein sets.Western blot assay was used to verify the expression of BDH2 in cells.Results:In A2780 cells,196 different proteins were found between the MET group and the control group.In SKOV3 cells,226 different proteins were found between the MET group and the control group.Fisher's Exact Test was used to analyze the GO function enrichment of different expressed proteins.After treated with MET,in A2780 cells,iron ion homeostasis,T cell proliferation,lymphocyte proliferation,cation transport,mononuclear cell proliferation and other important biological processes changed significantly.Ferric iron binding,platelet derived growth factor,platelet activating factor receptor activity,molecular carrier activity,platelet-derived growth factor-activated receptor activity and other molecular function changed significantly.Significant changes occurred in locational proteins,including cytosolic large ribosomal subunit,cytosolic ribosome,ferritin complex,intracellular Ferritin complex,cytosolic part,etc.After treated with MET,in SKOV3 cells,mitochondrial translational elongation,mitochondrial translational termination,translational termination,mitochondrial translation,translational elongation and other important biological processes changed significantly.Some molecular function,such as structural constituent of ribosome,structural molecule activity,cofactor binding,iron-sulfur cluster binding,metal cluster binding and some localization proteins,such as organellar ribosome,mitochondrial ribosome,mitochondrial large ribosomal subunit,organellar large ribosomal subunit,large ribosomal subunit changed significantly.KEGG pathway enrichment analysis results showed that after treated with MET,mineral absorption,the bile secretion,ferroptosis,ribosome,tuberculosis,and other important pathways in A2780 cells changed dramatically.While ribosome,complement and coagulation cascades,porphyrin and glucose metabolism,tyrosine metabolism,staphylococcus aureus infection and other important pathways changed significantly.Western blot confirmed that the expression of BDH2 in ovarian cancer cells A2780 and SKOV3 was increased after being treated with MET.Conclusions:1.The mechanism of MET on different ovarian cancer cells is different.2.MET inhibits ovarian cancer cell A2780 mainly by affecting intracellular iron homeostasis.3.Metformin inhibits ovarian cancer cells SKOV3 mainly by affecting the function of mitochondria.