Synergy Between Chidamide And Cisplatin In Killing Ovarian Cancer Cells Via Targeting Energy Metabolism And Metabolomics-based Analysis On Chemoresistance Of Ovarian Cancer Cells

Among gynecological tumors,Ovarian cancer（OC）has the highest mortality rate due to its characteristics such as late diagnosis,short-term recurrence,easy chemotherapy and drug resistance^[1,2].At present,the standard treatment for ovarian cancer includes tumor cell reduction,platinum combined with paclitaxel chemotherapy according to the stage,and maintenance therapy of PARPi monotherapy/combined bevacizumab in some patients according to the clinical stage,initial therapeutic drug use and gene status of the patients^[3].Although the standard first-line treatment of OC patients has prolonged the progression-free survival of some patients or delayed recurrence,the recurrence of OC is still difficult to avoid^[2,4].Currently,70%of EOC patients relapse within three years of initial treatment^[2].With the increase in the number of relapses and the shortening of the interval,patients will eventually change from chemotherapy sensitivity to drug resistance,and drug resistance is the key factor leading to treatment failure^[4,5],So far,there is no effective treatment plan for patients with chemotherapy resistance.In this study,OC was investigated from two perspectives of synergistic cisplatin killing OC cells and metabolomic changes of drug-resistant cells.The first part mainly discusses the killing of ovarian cancer cells by cisplatin in coordination with sidarbenamine through targeted energy metabolism,and the second part discusses the non-targeted metabolomics studies of drug-resistant ovarian cancer cells.To provide new ideas and strategies for the treatment and overcoming drug resistance of OC patients.Part 1 Synergy between Chidamide and cisplatin in killing ovarian cancer cells via targeting energy metabolismBackground:Ovarian cancer（OC）is the 8th leading cause of cancer death in women worldwide,with the highest mortality rate among gynecological tumors,and its pathological type is mainly cutaneous（about 90%）.In most Epithelial ovarian cancer（EOC）,epigenetic changes are evident and overexpression of histone deacetylases（HDACs）is an important manifestation.histone deacetylase inhibitor（HDACi）plays an antitumor role by altering histone deacetylase in normal and transformed cells,disrupting the cycle of cancer cells and activating apoptosis.Recent studies have shown that HDACi can affect proliferation by acting on single or multiple enzymes in the glycolytic and oxidation pathways.In vivo,in vitro and clinical trials have shown that HDACi may be a promising antitumor drug,which has been shown to have anticancer effects on OC.Chidamide（Chi）is a novel benzoamide HDAC inhibitor that can selectively inhibit HDAC1,2,3,and 10.Chi is currently undergoing clinical trials in the United States and China for the treatment of a variety of solid tumors,including OC.Although previous in vivo and in vitro experimental studies have shown that the combined treatment of HDACi and carboplatin has a synergistic anti-tumor effect,there are insufficient studies on the effect and mechanism of Chi on OC,and it is not clear whether the combined treatment of Chi and cisplatin（CDDP）,the first-line chemotherapy drug of OC,has a synergistic effect.More basic research is needed to support clinical trials.This study further explored the effects of Chi and CDDP alone or in combination on proliferation,apoptosis and metabolism of ovarian cancer.Using ovarian cell lines A2780（BRCA wild type）and COV362（BRCA mutant）as models,the effects of Chi and CDDP alone and combined in vivo and in vitro on the growth of ovarian cancer cells and their mechanisms were observed.Based on glycolysis and mitochondrial pressure changes,the effects of Chi and CDDP alone and combined in vitro on the energy metabolism of ovarian cancer cell line A2780 and COV362 were studied.It was found that the combined treatment of Chi and CDDP regulates the transcription of metabolic enzymes,and the combined treatment of CDDP and Chi specifically enhances the expression of PDK4 and inhibits OXPHOS.Further studies found that the expression of PDK4 induced metabolic reprogramming of OC cells,affected OC cell cloning,migration,invasion and sensitivity to Chi and CDDP,and provided new ideas and strategies for the treatment of OC patients.Objectives1.Determine whether Chi+CDDP has a synergistic effect on OC cells;To explore the effects of Chi and CDDP on the proliferation and apoptosis of OC cells,and further verify in vivo;2.To clarify the effects of Chi and CDDP alone and in combination on OC cell metabolism;3.Explore the effects of Chi and CDDP combination on the transcription of metabolic enzymes in OC cells,and find specific changes in the transcription and translation expression of PDK4;4.To explore the effects of PDK4 on the biological function and metabolism of OC cells;5.In vivo experiments verified the effects of PDK4 on OC cell proliferation and drug sensitivity.Method1.The inhibitory effects of CDDP and Chi on A2780 and COV362 were detected by CCK-8 method,and the cell inhibitory rate and dose concentration were mapped,and the respective IC50 values were obtained;2.Different low concentrations of CDDP and Chi were selected for the combined experiment,and the Q value of the combined drug was calculated using the gold formula,and the effect of the combined drug of CDDP and Chi on OC cells was judged according to the Q value;3.Agilent Seahorse XF energy metabolism detection technology was used to detect the changes in the glycolytic pressure and mitochondrial pressure of A2780 and COV362 cells after CDDP and Chi alone or combined;4.Western Blot technique was used to detect the expression levels of mitochondrial damage,apoptosis and glycometaboly-related proteins in A2780 and COV362 cell after CDDP and Chi alone or combined;5.Used q-PCR technology to detect the m RNA expression of glycometabolism related proteins in OC cells after CDDP and Chi alone and combined;6.Lentivirus infection was used to knock down/overexpress PDK4 in OC cells,Agilent Seahorse XF energy metabolism detection technology was used to detect the effect of PDK4 expression on energy metabolism of OC cells;7.Explore the effects of PDK4 on OC cell cloning,migration,invasion and apoptosis;8.Western Blot was used to detect the expression levels of proteins related to glucose metabolism,Akt-m TOR signaling pathway and apoptosis pathway after PDK4 knockdown/overexpression in OC cells;9.The OC transplanted tumor model was established by subcutaneously transplant A2780-OE-PDK4,A2780-KD-PDK4 cells into BALB/c female mice,and drug treatment was given randomly,and the survival time of mice in each group was observed after drug withdrawal.Results1.Chi and CDDP can inhibit the proliferation of ovarian cancer cells;Chi combined with CDDP has synergistic effect on proliferation inhibition and apoptosis of ovarian cancer cells.In vivo experiments showed that Chi combined with CDDP could prolong the survival of OC implanted tumor model mice;2.Chi combined with CDDP inhibited energy metabolism of OC cells,significantly reducing glycolysis and mitochondrial metabolism;3.Chi combined with CDDP activated mitochondrial apoptosis pathway,decreased the expression of glycolytic pathway protein,and down-regulated the expression of p-m TOR and c-Myc;4.Chi combined with CDDP affects the transcription of metabolic enzymes,enhances the expression of PDK4 and inhibits OXPHOS;5.Overexpression of PDK4 up-regulated energy metabolism of OC cells and increased glycolytic capacity;Knocking down PDK4 down-regulates the expression of multiple glucose metabolizing proteins in OC cells,decreases the ability of energy metabolism,and significantly reduces the ability of glycolysis.The changes of OXPHOS vary from cell to cell,which may be related to BRCA mutation;6.Overexpression of PDK4 enhanced the cloning,invasion and migration ability of OC cells;Knockdown of PDK4 can weaken the cloning,invasion and migration ability of OC cells;7.Overexpression of PDK4 enhanced CDDP resistance of OC cells in vitro and in vivo,and down-regulated mitochondrial apoptosis pathway;8.Overexpression of PDK4 can activate the Akt-m TOR signaling pathway in OC cells;In PDK4 knockdown cells,p-m TOR was down-regulated and LC3Ⅱ/LC3Ⅰwas up-regulated.Conclusion1.Chi combined with CDDP can synergistically inhibit proliferation and promote apoptosis of OC cells in vivo and in vitro;2.Chi combined with CDDP decreased the glycolytic ability of OC cells,which may be related to the down-regulation of glycolytic related proteins,p-m TOR and c-Myc expression;3.Chi combined with CDDP affects the transcription of metabolic enzymes,enhances the expression of PDK4 and inhibits OXPHOS;The apoptosis of tumor cells was induced by activating mitochondrial apoptosis pathway.4.The expression of PDK4 affects the ability of OC cells to clone,migrate and invade in vitro,as well as the sensitivity of cells to CDDP chemotherapeutic drugs and the CDDP-induced mitochondrial apoptosis pathway.5.Knockdown of PDK4 expression inhibited the expression of glycolytic rate-limiting enzyme,inhibited energy metabolism,and significantly weakened the glycolytic ability of OC cells;Overexpression of PDK4 resulted in increased energy metabolism and glycolytic ability of OC cells.The changes of OXPHOS vary from cell to cell,which may be related to BRCA mutation.6.The regulation of biological function of OC cells by PDK4 may be related to PI3K/Akt signaling pathway.Part 2 Metabolomics-based study for chemoresistance in ovarian cancer cellsBackground: Ovarian cancer（OC）is the third most common malignancies in the female reproductive system,but it is the deadliest malignancies in the female reproductive system.At present,the standard treatment plan proposed by the National Comprehensive Cancer Network（?）（NCCN（?））for OC is tumor cell reduction,and platinum-based systemic periodic chemotherapy combined with paclitaxel is given for 3-6 courses according to postoperative pathological stage.According to the gene status and clinical stage of the patients,whether the patients were combined with vascular inhibitors during periodic chemotherapy,and other comprehensive factors,poly-ADP-ribose polymerase inhibitor（PARPi）monotherapy/combined beizumab maintenance therapy was given after chemotherapy.Even after standard treatment such as surgery,chemotherapy,and targeted drug maintenance therapy,OC relapse is still difficult to avoid.Relapse and drug resistance are the main causes of treatment failure and high mortality in OC patients,but the mechanism leading to relapse and drug resistance is still unclear.A signature feature of cancer cells is the ability to reprogram their metabolism to maintain a favorable environment for tumor growth.OC is a heterogeneous tumor with different pathological types showing different metabolic characteristics.For drug-resistant ovarian cancer,the metabolic changes are more complex,and the metabolic pathway plays an important role in the growth,drug resistance and metastasis of cancer cells.Therefore,to reveal the molecular mechanism of metabolic reprogramming in OC resistant cells is one of the urgent problems to be solved.It is of great significance for the treatment and prognosis of OC.Metabolome,as another emerging discipline after genomics,transcriptomics and proteomics,refers to the total number of metabolites that exist in cells,tissues,organs or organisms and have a wide range of functions.Metabolites are not only the products and substrates of cell metabolism,but also reflect the changes in cell function related to tumor occurrence and development,such as cell proliferation,apoptosis,and metastasis.Therefore,changes in metabolite levels can be used as diagnostic and prognostic markers,as well as therapeutic targets.In this study,the drug-resistant cell models of ovarian cancer cell A2780 against Cisplatin（CDDP）resistant cell ARC,Paclitaxel（PTX）resistant cell ARP and Olaparib/Ola resistant cell ARO were constructed.ultra performance liquid chromatography-quadrupole time-of flight mass spectrometry,Ultra performance liquid chromatography-Quadrupole Time-of Flight mass spectrometry,UHPLC-Q-TOF-MS was used for non-targeted metabolomics detection and bioinformatic functional analysis of differential metabolites to explore the metabolic mechanism of ARC,ARP and ARO cell drug resistance,providing new ideas and strategies for reversing first-line chemotherapy drugs and targeting drug resistance maintenance for ovarian cancer.Objectives: 1.Ovarian cancer cell models of A2780 cisplatin resistant cell ARC,paclitaxel resistant cell ARP,and Ola resistant cell ARO were constructed,and the metabolic mechanism of drug resistance of ARC,ARP and ARO resistant cell line was explored on this basis;2.To determine the effects of CDDP exposure on metabolites of A2780 and ARC cell;Effects of PTX exposure on metabolites of A2780 and ARP cell;Effects of Ola exposure on metabolites of A2780 and ARO cell;3.Explore the effects of CDDP,PTX and Ola on differential metabolite enrichment pathways in A2780 parents and drug-resistant cell lines,and analyze metabolism markers of chemoresistance,pathways and therapeutic pathways that potentially reverse resistance.Method: 1.The A2780 cell resistant to first-line chemotherapy drugs of OC was constructed by gradient increasing concentration,and respectively are ARC,ARP,ARO;2.The IC50 of CDDP for A2780 and ARC,PTX for A2780 and ARP,Ola for A2780 and ARO cell were detected by CCK8;The resistance and stability of drug-resistant cells were verified by cell proliferation,growth curves and cloning experiments;3.Flow cytometry was used to detect the apoptosis ratio of A2780 and ARC cells treatment with CDDP;Apoptosis rate of A2780 and ARP treated with PTX;Apoptosis ratio of A2780 and ARO cells treatment with Ola;4.UHPLC-Q-TOF-MS technique was used to detect the differential metabolites between A2780,ARC,CDDP treated A2780（A2780-CDDP）and ARC（ARC-CDDP）groups.The bioinformatics function of different metabolites ware analyzed;5.UHPLC-Q-TOF-MS technique was used to detect the differential metabolites between A2780,ARP,PTX treated A2780（A2780-PTX）and ARP（ARP-PTX）groups.The bioinformatics function of different metabolites ware analyzed;6.UHPLC-Q-TOF-MS technique was used to detect the differential metabolites between A2780,ARO,Ola treated A2780（A2780-Ola）and ARO（ARO-Ola）groups.The bioinformatics function of different metabolites ware analyzed.Results: 1.Drug-resistant cell ARC,ARP and ARO were successfully constructed,and the proliferation of ARC,ARP and ARO cell lines was slowed down;Inhibition of proliferation and promotion of apoptosis induced by CDDP,PTX and Ola were decreased;2.Analysis of the differential metabolites of ARC vs A2780 and ARC-CDDP vs A2780-CDDP,identification added interaction analysis,combined with the results of KEGG analysis,the markers of cisplatin resistance of A2780 were identified,they are Udp-n-acetylglucosamine,L-citrulline,DL-proline,Udp-galactose,N-（octagecyl）-sphingo4-enpurine-1-phosphate choline,valine.Differential metabolites enrichment showed that CDDP resistance pathways include Alanine metabolism,Central carbon metabolism,Pyrimidine metabolism,Amino sugar and nucleotide sugar metabolism,Glutamic acid metabolism,Arginine metabolism,Aspartate metabolism,m TOR signaling pathway,ABC transporters.3.KEGG analysis the differential metabolites of ARC vs A2780 and ARC-CDDP vs A2780-CDDP.Only in the CDDP exposed group were abnormally enriched pathways: Glyoxylic acid,Dicarboxylic acid metabolism,2-oxy-carboxylic acid metabolism pathway,Metabolism of Phenylalanine,Glycine,Serine,Threonine,Tyrosine and Tryptophan,Proline metabolism and Sphingolipid signaling pathways.4.Analysis of the differential metabolites of ARP vs A2780 and ARP-PTX vs A2780-PTX,identification added interaction analysis,combined with the results of KEGG analysis,the markers of PTX resistance of A2780 were identified they are Ile-Pro,deoxyinosine,deoxythoryside,Myo-inositol and niacinamide.Differential metabolite enrichment shows that paclitaxel resistance pathways include Alanine metabolism,glutamic acid metabolism,Aspartic acid metabolism,Pyrimidine metabolism,Galactose metabolism,Phenylalanine metabolism,ABC transporter,and Neural active ligand-receptor interaction.5.KEGG analysis differential metabolites of ARP vs A2780 and ARP-PTX vs A2780-ptx.Only in the PTX exposed group were abnormally enriched pathways: Pentose phosphate pathway,Amino sugar and nucleotide sugar metabolism;Glycine,Serine and Threonine metabolism,Arginine biosynthesis,Purine metabolism and Sphingolipid signaling pathways.6.By analyzing the differential metabolites of ARO vs A2780 and ARO-Ola vs A2780-Ola,identification added interaction analysis,combined with the results of KEGG analysis,the markers of Ola resistance of A2780 were identified they are N-acetylaspartylglutamic acid,N-Acetyl-Asp-Glu,pyridine,Acetylcholine,L-palmitoylcarnitine,Myo-inositol,niacinamide.Differential metabolite enrichment shows that paclitaxel resistance pathways include Central carbon metabolism in cancer,Alanine metabolism,Glutamic acid metabolism,Threonine metabolism,Aspartic acid metabolism,Serine metabolism,Choline metabolism in cancer,Glycerophospholipid metabolism,Neuroactive ligand-receptor interaction,and ABC transporter.7.KEGG analysis the differential metabolites of ARO vs A2780 and ARO-Ola vs A2780-Ola.The pathways that were abnormally enriched only in the Ola-exposed group were Starch and sucrose metabolism,Galactose metabolism,2-oxy-carboxylic acid metabolism pathway,Taurine and hypotaurine metabolism,Pantothenate and Co A biosynthesis.Conclusions:1.Drug resistant cell ARC,ARP and ARO shared metabolic markers of drug resistance,Myo-inositol and niacinamide;shared drug resistance pathways include Central carbon metabolism in cancer,Alanine metabolism,Glutamic acid metabolism,Aspartic acid metabolism,ABC transporter pathway and Neural active ligand-receptor interaction pathway.Shared potential reversal of drug resistance pathways 2-oxycarboxylic acid metabolism pathway,Glycine metabolism,Sphingolipid signaling pathway,Serine metabolism,Threonine metabolism.The difference enrichment in 2 or 3 drug resistant cell lines indicated that there may be some common resistance mechanism of different drug resistance.2.Specific pathways for CDDP resistance of A2780 are include m TOR signaling pathway,Amino sugars and nucleotide sugars metabolism,Glutamic acid metabolism,Arginine metabolism,Aspartic acid metabolism;Potential pathways to reverse CDDP resistance of A2780 include metabolism of Phenylalanine,Tyrosine,Proline,Tryptophan,Glyoxalic acid and Dicarboxylic acid;3.Specific pathways for PTX resistance of A2780 include galactose metabolism and phenylalanine metabolism;Potential pathways to reverse PTX resistance of A2780 include Amino sugar and nucleotide sugar metabolism,Arginine metabolism,and Pentose phosphate pathway,Purine metabolism;4.Specific pathways for Ola resistance of A2780 include Choline metabolism in cancer,Glycerol phospholipid metabolism,Serine metabolism and Threonine metabolism;Potential pathways for reversing Ola resistance of A2780 include Galactose metabolism,Starch and sucrose metabolism,Pantothenate and Co A biosynthesis,Taurine and hypotaurine metabolism...