| Ovarian cancer was the most challenging malignant tumor among gynecological oncology,and the pathogenesis was insidious and the symptoms were non-specific,causing most patients were developed to the advanced stage when diagnosed.At present,the standard treatment for ovarian cancer is surgery assisted by chemotherapy,radiation therapy or biological therapy.However,the recurrence rate of ovarian cancer after treatment was 70-80%,and the 5-year survival rate was less than 40%.Traditional chemotherapeutics such as cisplatin targeted DNA and induced apoptosis of ovarian cancer cells by interfering with DNA homeostasis,but they were prone to drug resistance.Although,studies have found that cisplatin resistance was related to mitochondrial function,in addition to energy production,cell metabolism,redox balance,and apoptosis,mitochondria also played a key role in intracellular signal transmission.Mitochondrial DNA only encodes 13 proteins,all other mitochondrial proteins are encoded by nuclear DNA,including the classic antioxidant pathway SIRT3-SOD2,all nuclear DNA encodes proteins enter the mitochondria through the mitochondrial import system for proper positioning and assembly to function.In addition to endoplasmic reticulum and mitochondrial stress,cytoplasmic proteins,as proteins communication platform for mitochondria and other organelles,play a special role in maintaining cell proteostasis.As a conservative molecular chaperone,cytoplasmic heat shock protein 90(HSP90)precisely controls the processes of protein synthesis,folding,conformational stability and degradation,and plays the dual role of executor and coordinator in these processes.Current studies have found that molecular chaperones such as HSP90 and HSP70 are highly expressed in lung cancer,liver cancer and ovarian cancer,and they are believed to be closely related to tumor cell proliferation,metastasis and drug resistance.HSP90 can directly interact with translocase of the outer membrane,TOM70,transporting client molecules into the mitochondria through the mitochondrial transport system,and then participates in the regulation of cell stress,redox,and apoptosis.Therefore,furtherly exploring the relationship between HSP90-mediated cytoplasmic protein homeostasis and mitochondrial function,it may provide a new strategy for increasing the sensitivity of cisplatin and other chemotherapeutic drugs.Therefore,this experiment used cisplatin high-sensitivity ovarian cancer A2780cells and low-sensitivity ovarian cancer SKOV3 cells as subjects,and HSP90inhibitor SNX2112 used to construct the model of proteostasis imbalance,and focusing on the mitochondrial transport system.Combining proteomics and bioinformatics to analyze the coordination mechanism of mitochondria,and explore the effect of cytoplasmic proteostasis on mitochondrial redox/energy production,finally clarified the role and mechanism of mitochondria in the formation of cisplatin resistance in ovarian cancer cells.Methods:1.To detect the difference in the sensitivity to cisplatin,as well as the differences in ATP content,ROS level and protein expression between ovarian cancer SKOV3cells and A2780 cells.Different doses of cisplatin were used to treat ovarian cancer SKOV3 cells and A2780 cells,cell viability was determined by CCK-8 kit;ATP content,ROS level and differences in protein expression between SKOV3 cells and A2780 cells were detected by ATP and ROS detection kit and Western blot.2.To detect the difference in the sensitivity to HSP90 inhibitor SNX2112between ovarian cancer SKOV3 cells and A2780 cells.Different doses of SNX2112were used to treat ovarian cancer SKOV3 cells and A2780 cells,and the cell viability was determined by CCK-8 kit.After clarifying the dose of SNX2112,the apoptosis rate was detected by flow cytometry;the expression of AKT1 was detected by Western blot to determine the specificity and inhibitory effect of SNX2112.3.To observe the effect of HSP90 inhibitor SNX2112 on the mitochondrial ultrastructure of ovarian cancer SKOV3 cells and A2780 cells.The ovarian cancer SKOV3 cells and A2780 cells were treated with 200 n M SNX2112 for 24 h,and the changes of mitochondrial ultrastructure were observed by transmission electron microscope.4.To detect the effect of HSP90 inhibitor SNX2112 on the mitochondrial protein import capacity of ovarian cancer SKOV3 cells and A2780 cells.Ovarian cancer SKOV3 cells and A2780 cells were treated with 200 n M SNX2112 for 12 h,24 h and48 h.Mitochondria were separated and purified,and mitochondrial import system proteins were analyzed by proteomics and GO enrichment(including:TOM system,TIM system and PAM system),and finally the expression of theses proteins was verified by Western blot.The mitochondrial membrane potential(MMP)was marked by JC-1 staining,and the MMP changes were detected by flow cytometry.5.To detect the effect of HSP90 inhibitor SNX2112 on energy production/redox in ovarian cancer SKOV3 cells and A2780 cells.Ovarian cancer SKOV3 cells and A2780 cells were treated with 200 n M SNX2112 for 12 h,24 h and 48 h.ATP and ROS detection kits were used to detect changes in intracellular ATP content and ROS levels,and glucose and lactate detection kits were used to detect glucose consumption capacity and lactic acid secretion capacity in ovarian cancer cells.6.To detect the effect of HSP90 inhibitor SNX2112 on mitochondrial import of respiratory chain complex in ovarian cancer SKOV3 cells and A2780 cells.Ovarian cancer SKOV3 cells and A2780 cells were treated with 200 n M SNX2112 for 12 h,24 h and 48 h.mitochondria were separated and purified.The expression changes of mitochondrial respiratory chain complex subunits were analyzed by proteomics and KEGG signal pathway enrichment,and Western blot was used to verify the expression of these proteins.7.To detect the effect of HSP90 inhibitor SNX2112 on oxidoreductase activity relative proteins mitochondrial import in ovarian cancer SKOV3 cells and A2780cells.Ovarian cancer SKOV3 cells and A2780 cells were treated with 200 n M SNX2112 for 12 h,24 h and 48 h.Mitochondria were separated and purified.The expression changes of oxidoreductase activity relative proteins at the mitochondrial level were analyzed by proteomics and GO enrichment,and Western blot was used to verify the expression of SRIT3/SOD2.8.To detect the effect of HSP90 inhibitor SNX2112 on SIRT3 activity of ovarian cancer SKOV3 cells and A2780 cells.NAD~+detection kit was used to analyze the content of NAD~+in ovarian cancer cells,and CCK-8 viability detection kit was used to detect the effect of SIRT3 specific inhibitor 3-TYP and overexpression of SIRT3on the viability of ovarian cancer cells.The expression changes of long-SIRT3 and short-SIRT3 were detected by Western blot.Results:1.The sensitivity of ovarian cancer SKOV3 cells to cisplatin was significantly lower than that of A2780 cells.The ATP content and mitochondrial membrane potential of SKOV3 cells were significantly higher than that of A2780 cells,while ROS levels,HSP90?,HSP90?,SIRT3,SOD2,TOM-TIM system protein expression are significantly lower than A2780 cells,indicating that the difference in cisplatin sensitivity of SKOV3 cells and A2780 cells may be related to these factors.2.The sensitivity of ovarian cancer SKOV3 cells to HSP90 inhibitor SNX2112is similar to that of cisplatin,which is significantly lower than that of A2780 cells.Further observation of the mitochondrial ultrastructure shows that SKOV3 cells have lighter extent of mitochondrial damage,with excellent mitochondrial cristae content and integrity than A2780 cells,indicating that the difference in sensitivity of SKOV3cells and A2780 cells to SNX2112 and cisplatin may be related to mitochondrial function.3.The mitochondrial protein import capacity was further detected.The proteomics and GO enrichment analysis results showed that after treatment with the HSP90 inhibitor SNX2112,the mitochondrial transport system proteins expression of SKOV3 cells was significantly increased,and the protein import capacity was significantly enhanced,while the A2780 cells mitochondrial transport system proteins expression and transport capacity were significantly reduced;and the increase in mitochondrial membrane potential of SKOV3 cells was significantly higher than that of A2780 cells;indicating that mitochondrial protein import capacity is the key factor to determine the difference in mitochondrial function and drug sensitivity.4.The HSP90 inhibitor SNX2112 can significantly increase the level of ROS in ovarian cancer cells.In the basal state,the ROS level of A2780 cells is significantly higher than that of SKOV3.After SNX2112 treated for 48 hours,the level of ROS in SKOV3 cells is equivalent to the basic level of A2780,indicating that SNX2112 can improve the oxidative stress level to reach the lethal threshold and induces the apoptosis of ovarian cancer cells,but the difference in the buffering capacity to oxidative stress between SKOV3 cells and A2780 cells leads to differences in apoptosis.5.The mitochondrial function was further detected,and the results showed that the ATP content in SKOV3 cells did not change significantly with the extension of SNX2112 treatment time,while the ATP content in A2780 cells decreased significantly;the consumption of glucose increased in the early stage and decreased in the late stage in both ovarian cancer cells;SKOV3 Cell lactic acid secretion continued to decrease,and A2780 cell lactic acid secretion firstly decreased and then increased,indicating that energy metabolism of SKOV3 cells shifted to more efficient oxidative phosphorylation after SNX2112 treatment,while A2780 shifted to less efficient glycolysis.The change in mitochondrial function may be primary cause of the difference in drug resistance of the two types of ovarian cancer cells.6.The results of proteomics and bioinformatics analysis showed that the function of high-expressed protein in mitochondria of SKOV3 cells mainly concentrated on oxidative phosphorylation and the tricarboxylic acid cycle pathway after treatment with HSP90 inhibitor SNX2112;further screening and Western blot results showed:the expression of mitochondrial respiratory chain complex in SKOV3 cells increased significantly,while the A2780 cells showed the opposite trend.It indicated that SKOV3 cells maintain energy production/redox homeostasis by improving nuclear-mitochondrial coordination,furtherly increasing the mitochondrial import and assembly of the subunits of the respiratory chain complex.7.With the extension of the treatment time of HSP90 inhibitor SNX2112,proteomics and bioinformatics analysis results showed that he function of high-expressed protein in mitochondria of SKOV3 cells mainly concentrated on the redox pathway,while A2780 cells showed the opposite trend;further screening and Western blot results showed:The expression of SIRT3 and SOD2 at the overall level of the two ovarian cancer cells were significantly increased;while the expression of SIRT3 and SOD2 in the mitochondria of A2780 cells decreased continuelly,and the expression of SIRT3 and SOD2 in the mitochondria of SKOV3 cells increased continuelly;indicating the synergy of SIRT3 and SOD2 mitochondrial import was the key factor to determine the drug resistance of ovarian cancer cells.8.The NAD~+content in SKOV3 cells was significantly higher than that in A2780 cells under the basal state.After SNX2112 treatment,the NAD~+content in SKOV3 cells decreased,but there was no statistical significance,while NAD~+in A2780 cells was significantly reduced;SKOV3 cells was more sensitive to SIRT3inhibitors than A2780 cells;Overexpression of SIRT3 did not reduce the sensitivity of ovarian cancer cells to SNX2112,Western blot results showed that overexpression of SIRT3 failed to enter the mitochondria to play a role,while protein-protein interaction analysis SIRT3 and SOD2 can directly interact with the respiratory chain complex,which further confirmed that imported the respiratory chain complex and SIRT3/SOD2 antioxidant system proteins into mitochondria synergistically was the core mechanism for coordinating energy production/oxidative stress and determining the drug resistance of ovarian cancer cells.Conclusion:1.The sensitivity of cisplatin-resistant SKOV3 cells and cisplatin-sensitive A2780 cells to HSP90 inhibitor SNX2112 was consistent with that of cisplatin,suggesting that the balance of energy production(ATP production)and oxidative stress(ROS production)of cisplatin-resistant ovarian cancer cells may be related to the decreased sensitivity to HSP90 inhibitors.2.Mitochondrial proteomics and bioinformatics analysis found that cisplatin-resistant SKOV3 cells have a strong protein balance capacity.Cell experiments have verified that the mitochondrial protein import ability may be a primary cause of cisplatin resistance.3.Cisplatin-resistant SKOV3 cells have relatively strong antioxidant capacity,and the mitochondrial import of antioxidant proteins such as NAD+/SIRT3/SOD2may be related to the balance of energy production(ATP production)and oxidative stress(ROS production)in cisplatin-resistant ovarian cancer cells.4.The cross-talk between nucleus and mitochondria coordinated the mitochondrial import of respiratory chain complex and antioxidant-related proteins.The balance between energy production and redox of ovarian cancer cells established by mitochondrial import may be one of the mechanisms leading to cisplatin resistance in ovarian cancer. |
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