ANAX1 Involved In Paclitaxel Resistance In Ovarian Cancer Via The PI3K/AKT/PGC1α Axis

Background: Ovarian cancer is a malignant tumor with a high degree of heterogeneity.It is the most common malignant tumor of the female reproductive system with the highest fatality rate.Paclitaxel is the first-line chemotherapeutic drug for the treatment of ovarian cancer.It is currently widely used.However,the resistance of ovarian cancer cells to paclitaxel severely hinders the effect of chemotherapy,resulting in a decrease in patient survival.The drug resistance mechanism of paclitaxel is complex and has not yet been fully elucidated.The PI3K/AKT/mTOR signaling pathway plays a key role in mediating cell proliferation and survival,and is one of the most commonly affected pathways in human cancers.Studies have suggested that its excessive activation is involved in the formation of platinum and paclitaxel resistance.As a key protein of mitochondrial biosynthesis and energy metabolism,peroxisome proliferative activated receptor（peroxisome proliferative activated receptor,gamma,coactivator 1 alpha,PGC1α）,its expression increases will lead to an increase in antioxidant enzymes and The reduction of apoptosis induced by ROS,which promotes the migration and invasion of cancer cells,has been proved to be related to the formation of cisplatin resistance in ovarian cancer.Recently,it has been reported in the literature that activation of the PI3K/AKT signaling pathway will promote the up-regulation of PGC1α.Annexin A1（annexin A1,ANXA1）is a type of calcium phospholipid binding protein,which is widely present in cells.It participates in a variety of important life processes by binding to formylpeptide receptor（FPR1/FPR2）,such as Cell signal transduction,differentiation,apoptosis,etc.,ANXA1 can induce AKT phosphorylation by activating PI3 K.In recent years,it is generally believed that ANXA1 is related to the occurrence of ovarian cancer and chemotherapy resistance,and can be used as a biomarker of ovarian cancer drug resistance and prognosis.However,specific mechanism of ANXA1 affecting chemotherapy resistance is still unclear,and further exploration is needed.Objective: Explore the biological mechanism of ANXA1 affecting paclitaxel resistance in ovarian cancer cells（A2780）and ovarian cancer paclitaxel-resistant cells（A2780/PA）,and discover new biomarkers and therapeutic targets for paclitaxel resistance in ovarian cancer cells.Provide a new strategy for the treatment of ovarian cancer patients with resistance to paclitaxel.Method: 1.Treat ovarian cancer A2780 cells and ovarian cancer Paclitaxel-resistant A2780/PA cells with different concentrations of paclitaxel,after 24 hours of treatment,use MTT method to detect cell viability;2.Real-time Quantitative PCR（Real-time Quantitative PCR）technology was used to detect the mRNA levels of ANXA1 and PIK3R1 genes in A2780 and A2780/PA cells;Western Blot technology was used to detect differences in the expression of ANXA1,mitochondrial biosynthesis key protein PGC1α and downstream related proteins;3.At present,scholars generally believe that the active peptide Ac2-26,as a mimetic peptide of ANXA1,can bind to FPR and exert the function of ANXA1 in cells.Paclitaxel（0.00625 μg/mL）combined with Ac2-26（3 μM）treated A2780 cells for 24 h,MTT method was used to detect cell viability,flow cytometry was used to detect cell apoptosis rate,and Western Blot technology was used to detect changes in apoptosis-related proteins;4.At present,it is recognized that Boc2,as an FPR inhibitor,can block the binding of ANXA1 to the receptor and inhibit the function of ANXA1 in cells.Paclitaxel（10 μg/mL）combined with Boc2（10 μM）treated A2780/PA cells for 24 h,Use MTT method to detect cell viability,flow cytometry to detect cell apoptosis rate,Western Blot technology to detect changes in apoptosis-related proteins;5.After treating A2780 cells with paclitaxel（0.00625 μg/mL）combined with Ac2-26（3 μM）for 24 h,Western Blot technology was used to detect the protein expression of PI3 K,P-AKT and AKT;6.After treating A2780/PA cells with paclitaxel（10 μg/mL）combined with Boc2（10 μM）for 24 h,Western Blot technology was used to detect the protein expression of PI3 K,P-AKT and AKT;7.After paclitaxel（0.00625 μg/mL）combined with Ac2-26（3 μM）treated A2780 cells for 24 h,Western Blot technology was used to detect the expression changes of the key protein PGC1α and downstream related proteins in mitochondrial biosynthesis,and the multifunctional microplate reader was used to detect the level of ATP production;8.Paclitaxel（10 μg/mL）combined with Boc2（10 μM）or PI3 K inhibitor PKI-402（10 μM）was used to treat A2780/PA cells for 24 hours,and Western Blot technology was used to detect the expression of mitochondrial biosynthesis key protein PGC1α and downstream related proteins Change,the multifunctional microplate reader detects the level of ATP production.Treat ovarian cancer A2780 cells and ovarian cancer Paclitaxel-resistant A2780/PA cells with different concentrations of paclitaxel,and use MTT test to detect cell viability after 24 hours;Results: 1.The IC₅₀ of paclitaxel of A2780 is 0.664 μg/mL and the IC₂₀ is 0.00625 μg/mL;the IC₅₀ of paclitaxel of A2780/PA is 31.47 μg/mL,IC₂₀ is 10 μg/mL,and its drug resistance index Is 47.4.In the subsequent experiments,the dosage of the drug is selected as the concentration of paclitaxel corresponding to IC₂₀;2.Compared with A2780 cells,the mRNA levels of ANXA1 and PIK3R1 in A2780/PA cells increased significantly,and the expression of ANXA1,the key protein of mitochondrial biosynthesis PGC1α and downstream related proteins increased significantly;3.In A2780 cells,compared with the paclitaxel alone group,the cell activity of the Ac2-26 combined with paclitaxel group increased,the apoptosis rate decreased,and the expression of apoptosis-related protein Cleaved-caspase3 and the ratio of Bax/Bcl-2 decreased;4.In A2780/PA cells,compared with the paclitaxel alone group,the cell activity of Boc2 combined with paclitaxel increased,the apoptosis rate decreased,the expression of apoptosis-related protein Cleaved-caspase3 and the ratio of Bax/Bcl-2 increased;5.In A2780 cells,compared with the paclitaxel alone group,the PI3 K protein expression and P-AKT/AKT ratio increased in the Ac2-26 combined paclitaxel group;6.In A2780/PA cells,Boc2 combined with paclitaxel compared with the paclitaxel alone group The expression of PI3 K protein and the ratio of P-AKT/AKT in group cells decreased;7.In A2780 cells,compared with the paclitaxel alone group,the Ac2-26 combined with paclitaxel group increased the expression of the key protein PGC1α and downstream related proteins in mitochondrial biosynthesis,and increased the level of ATP production;8.In A2780/PA cells,compared with the paclitaxel alone group,the expression of the key mitochondrial biosynthesis protein PGC1α and downstream related proteins in the Boc2 combined with paclitaxel group and the PKI-402 combined with paclitaxel group decreased,and the level of ATP production decreased.Conclusion: 1.A2780/PA cells ANXA1,mitochondrial biosynthesis key protein PGC1α and downstream related proteins are highly expressed,and have stronger tolerance to paclitaxel,suggesting that ANXA1 and PGC1α-mediated mitochondrial biosynthesis may be related to paclitaxel resistance in ovarian cancer cells;2.ANXA1 can promote the activation of PI3K/AKT signaling pathway,inhibit cell apoptosis,and increase cell viability.The above results suggest that ANXA1 mediates the activation of PI3K/AKT signaling pathway through FPR,and increases the drug resistance of ovarian cancer cells to paclitaxel through the apoptosis pathway;3.ANXA1 promotes the expression of the key protein PGC1α and downstream related proteins in mitochondrial biosynthesis,and increases the level of mitochondrial energy metabolism,suggesting that ANXA1 mediates mitochondrial biosynthesis and mitochondrial energy metabolism through the PI3K/AKT signaling pathway,thereby affecting the paclitaxel resistance of ovarian cancer cells.Therefore,we believe that ANXA1 can cause paclitaxel resistance in ovarian cancer cells through the PI3K/AKT/PGC1α axis.