SIRT5 Promotes Cisplatin Resistance In Ovarian Cancer By Suppressing DNA Damage In A ROS-Dependent Manner Via Regulation Of The Nrf2/HO-1 Pathway

Objective:Globally,ovarian cancer ranked eighth in incidence and seventh in mortality among all cancers in females in 2018(WHO,http://gco.iarc.fr/today/home).Standard treatment for this disease involves surgery combined with chemotherapy.However,chemoresistance has become a major reason for poor outcomes in ovarian cancer Although emerging targeted therapies have improved the survival of chemoresistant ovarian cancer patients,their quality of life and overall survival are still limited due to the side effects associated with such drugs,and the 5-year survival rate for advanced-stage ovarian cancer is only 28.9%.Therefore,there is an urgent need to find specific biomarkers and clarify the molecular mechanism of drug resistance to improve the therapeutic efficacy of ovarian cancerSirtuins(SIRTs)are a family of intracellular enzymes that possess nicotinamide adenine dinucleotide(NAD+)-dependent deacetylase activity and share a highly conserved catalytic core domain.There are seven members(SIRT1-7)in mammals.Emerging evidence suggest that SIRTs play vital roles in tumorigenesis by regulating energy metabolism,DNA damage repair,genome stability,and various other cancer-associated cellular processes.It has been reported that SIRTs are closely associated with ovarian cancer.Presently,the dysregulated expression of SIRTs and their prognostic value have been partly reported in ovarian cancer and it is striking that even in the same tumor,the specic roles of individual SIRTs can be controversial,which may be partly ascribed to small sample sizes.A comprehensive analysis of the expression and mutation patterns and prognostic values of SIRTs in ovarian cancer based on large database analysis would enhance the understanding of their potential roles in ovarian cancer.Therefore,we conducted this study to investigate this phenomenonSIRT5 is a unique member of the SIRTs family,which possesses multiple enzymatic activities including NAD+-dependent histone deacetylase,potent lysine demalonylase,lysine desuccinylase,and lysine glutarylase activities.These specific enzymatic activities indicate that SIRT5 plays a crucial role in regulating multiple cellular metabolic processes such as glycolysis,the tricarboxylic acid cycle,fatty acid oxidation,nitrogen metabolism,and the pentose phosphate pathway.In addition,certain aspects of cancer biology,such as stress responses,apoptosis,and autophagy,can be regulated by SIRT5 Moreover,altered cellular metabolism has been recently identified as a hallmark of malignancy and emerging literature suggests that SIRT5 is involved in oncogenesis.For example,either the mRNA or protein expression of SIRT5 was found to be increased in non-small cell lung cancer(NSCLC),hepatocellular carcinoma colorectal cancer(CRC),Waldenstrom macroglobulinemia,and breast cancer,compared to the levels in matched normal tissues.Janus-faced roles of SIRT5 in cancer have also been described Specifically,the downregulation of SIRT5 was observed in head and neck squamous cell carcinoma,liver cancer,and endometrial carcinoma,which highlight the tumor-suppressive role of SIRT5.Furthermore,controversial roles for SIRT5 in drug resistance have been reported.SIRT5 facilitates NSCLC resistance to cisplatin,5-fluorouracil,and bleomycin.Moreover,SIRT5-positive cells in wild-type Kras CRCs were found to be resistant to either chemotherapeutic agents or cetuximab.However,a positive association between SIRT5 expression and complete response to neoadjuvant chemotherapy in triple-negative breast cancer patients was previously shown by analyzing data from the Gene Expression Omnibus DataSet.In addition,by analyzing the Oncomine online database,SIRT5 expression levels were found to be higher in chemotherapy-responders than in non-responders.Considering these discrepant findings,further studies are needed to explore the functions of SIRT5 in tumorigenesis and chemoresistance.Considering the diverse roles of SIRT5 in cancer biology and the results of above bioinformatics analysis(SIRT5 is overexpressed in ovarian cancer and is related to prognosis),we speculated that SIRT5 is a therapeutic target for ovarian cancer.To date,the role of this protein in ovarian cancer has not been elucidated.Therefore,in this context,we investigated the expression pattern of SIRT5 in both human ovarian cancer tissues and ovarian cancer cells.Furthermore,we reveal a potential role for SIRT5 in ovarian cancer cell growth and chemoresistanceMethods:1.The transcriptional expression patterns,prognostic values,and genetic alterations of seven SIRTs in ovarian cancer patients were investigated using a range of databases:Oncomine and Gene Expression Profiling Interactive Analysis(GEPIA),Kaplan-Meier Plotter,the Cancer Genome Atlas(TCGA),and cBioPortal.qRT-PCR and immunohistochemistry were used to confirm these findings.The protein-protein interaction networks of SIRTs were assessed in the String database.Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway were analyzed in Database for Annotation,Visualization,and Integrated Discovery(DAVID)2.SIRT5 staining was performed on an ovarian cancer and normal tissue microarray,which contained 90 tumor tissues and 10 normal ovarian tissues.Immunostaining was performed based on the ABC immunostaining protocol.Two independent investigators scored the microarray by evaluating the staining intensity and percentage of stained cells blindly and randomly.The staining intensity was scored from 0-3 as follows:0(negative),1(weak),2(moderate),and 3(strong).The percentage of positively stained cells was scored from 0 to 4 as follows:0(0%),1(1-25%),2(26-50%),3(51-75%),and 4(76-100%).The final multiplied scores ranged from 0-12 and SIRT5 expression was regarded as positive if the final score was?63.A2780,SKOV-3,and CAOV-3 human ovarian cancer cell lines were used in this study A2780 and CAOV-3 cells were cultured in DMEM supplemented with 10%fetal bovine serum(FBS).SKOV-3 cells were cultured in RPMI-1640 medium supplemented with 10%FBS.These cells were grown at 37? in a humidified atmosphere with 5%CO2Transient transfection was carried out using Lipofectamine 3000 reagent according to the manufacturer's instructions.A SIRT5 expression plasmid and the corresponding empty plasmid were used for SIRT5 overexpression and as a negative control,respectively Cells were transfected with SIRT5-siRNA and negative control siRNA for SIRT5 knockdown experiments.The cells were pretreated with 5 mM NAC for 2 h to inhibit ROS generation.ML385 was added to cells at a concentration of 5 ?M for 48 h in the presence of cisplatin to block the Nrf2 pathway4.Cell Counting Kit-8(CCK-8)assays were performed to assess cell proliferation and cell viability in vitro.For cell proliferation,the cells were seeded in 96-well plates at a density of 3,000 cells/well and incubated for 5 days.For cell viability,the cells were seeded in 96-well plates at a density of 5,000 cells/well and treated with the indicated concentration of cisplatin for 48 h or 5 days,changing the cisplatin-containing medium every 2 days.Next,10 ?l of CCK-8 reagent was added to each well,and the cells were incubated for 2 h.The absorbance value(OD)of each well was measured at 450 nm and the cell viability was calculated as follows:cell viability(%)=experimental group OD value/control group OD value × 100%.IC50 values(50%inhibition of surviving fraction)were calculated using GraphPad Prism 7.0 software5.Cells were plated in 6-well culture plates at 500 cells/well.After incubation for 14 days at 37? in a humidified atmosphere at 5%CO2,the cells were washed three times with PBS and stained with Giemsa solution.The number of colonies containing?50 cells was then counted under a microscope.Colony formation efficiency was calculated as colony numbers/500 × 100%6.The ROS levels induced by cisplatin in ovarian cancer cells were detected using the probe 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA),which can be oxidized by intracellular oxygen to dichlorofluorescein,a highly fluorescent compound.After exposure to the indicated concentration of cisplatin for 2,6,24,or 48 h,the cells were incubated with a final concentration of 10 ?M DCFH-DA in the dark for 20 min at 37?in a humidified atmosphere at 5%CO2,after which the cells were washed three times with cold PBS to remove excess fluorescent probe.The cells were then resuspended in 300?l of PBS and assessed for fluorescence intensity using a flow cytometer.The data were analyzed using FlowJo X 10.0.7 SoftwareIntracellular GSH levels were measured using a Total Glutathione Assay Kit according to the manufacturer's instructions.Briefly,the ovarian cancer cells were harvested and lysed in the protein removal solution S provided in the kit.After incubation for 5 min at 4?,the samples were centrifuged at 10,000 g for 10 min at 4?.The supernatant was treated with assay solution for 5 min at 25? and the absorbance at 412 nm was measured using a microplate reader.Intracellular GSH levels were quantified by interpolation on standard curves and relative GSH levels were calculated by normalization to the values obtained from A2780 cells7.Cells were plated in 20-mm culture plates,pretreated with the indicated concentrations of cisplatin for 24 h to observe ?-H2AX foci formation,washed with PBS three times,fixed with 4%paraformaldehyde for 15 min,and permeabilized in 0.1%Triton X-100 for 5 min.After blocking with 5%bovine serum albumin for 1 h at room temperature,the cells were incubated with primary antibodies against y-H2AX(dilution 1:200),SIRT5(dilution 1:200),or Nrf2(dilution 1:200)overnight at 4?.Then,TRITC/FITC-conjugated secondary antibody(dilution 1:1,000)was incubated with the cells for 2 h in the dark at room temperature,and the cells were stained with 4',6-diamidino-2-phenylindole(DAPI)for 5 min to visualize their nuclei.Images were captured using a fluorescence microscope or a confocal laser-scanning microscope.For quantification of ?-H2AX foci,5 random fields of cells from each slide were quantified by ImageJ software and foci containing?5 cells were considered positive8.Total protein was isolated from SIRT5 overexpressing or knockdown ovarian cancer cells and their corresponding controls,with or without cisplatin treatment.The cells were washed with ice-cold PBS three times and lysed in lysis buffer supplemented with a cocktail of proteinase inhibitors.Equal amounts of protein(60 ?g)from cell extracts were separated by 10%SDS-PAGE and transferred to 0.45-?m or 0.22-?m(the latter for?-H2AX)polyvinylidene fluoride(PVDF)membranes.Following blocking with 5%fat-free milk for 2 h at room temperature,the membranes were incubated with primary antibodies(anti-SIRT5,anti-actin,anti-Nrf2,anti-HO-1,anti-MnSOD/SOD2,anti-BRCA1,anti-histone H3,or anti-?-H2AX;dilution,1:1000)in blocking buffer overnight at 4?.Then,the membranes were washed three times in Tris-buffered saline with Tween 20 and incubated with secondary antibodies at a dilution of 1:2500 for 2 h at 37?.Immunoreactivity was detected using ECL with a BioImaging System.ImageJ software was used to evaluate the gray value of each band9.The GEPIA database(http://gepia.cancer-pku.cn/),a newly developed web-based tool,provides tumor versus normal differential expression analysis,correlation analysis based on the Cancer Genome Atlas,and genotype-tissue expression data.It was used to analyze the expression of SIRT5,Nrf2,and HO-1 in ovarian cancer and normal tissues and the correlation between SIRT5 and Nrf2,MnSOD,and BRCA1.All experiments were repeated at least three times and the data were expressed as the means±standard deviations.Statistical analysis was performed with GraphPad Prism 7.0 software Statistical significance was determined based on a Student's t-test or one-way ANOVA The ?2 test was used to determine the correlation between SIRT5 expression and clinicopathologic characteristics.P<0.05 was considered to denote a statistically significant differenceResults:1.By searching the Oncomine and GEPIA databases,we found that SIRTs were abnormally expressed in a variety of tumors.The expression levels of SIRT1-3 were significantly lower while SIRT5 was higher in ovarian cancer than normal tissues and it was significantly increased in stage ? tumors.The level of transcript expression varies slightly according to different pathological types and different databases.The above findings were verified by qRT-PCR and immunohistochemistry2.Using the Kaplan-Meier database,it was found that the decrease in SIRT1 and SIRT4 mRNA levels and the increase in SIRT2,3,6 and 7 expressions predict a good prognosis Overexpressed SIRT5 was associated with shorter progression-free survival but longer overall survival.The overall upregulation of SIRTs family mRNA expression was associated with poor prognosis in patients with ovarian cancer.According to the complex pathological type,pathological grade,clinical stage and TP53 mutation or not,combined with different observation indicators,the abnormal mRNA expression level of SIRTs showed good practical prognostic value3.To explore the genetic changes of SIRTs in patients with ovarian cancer,TCGA database and cBioPortal platform were used.The mutation rate of each SIRT member ranged from 1.4%to 10%.Among them,SIRT2,SIRT5 and SIRT7 had higher mutation rates than other members,and their mutation rates were 10%,8%and 5%,respectively There were many types of mutations,mainly amplification,but there was no obvious correlation between the mutation and the patient's prognosis4.Using the String database,a network consisting of 7 SIRT members and 20 proteins with obvious interaction was constructed.Among them,various cell metabolism-related proteins such as tumor protein 53(TP53),forkhead box O 1/3/4(FOXO 1/3/4),superoxide dismutase 2(SOD2),and histone post-transcriptional modification were related Genomic protein deacetylase 1/2/4(HDAC 1/2/4),E1A binding protein p300(EP300)and SUV39H1 were closely related to SIRTs.The Pearson correlation coefficient between SIRTs members was calculated using the GEPIA database,ranging from 0.073(SIRT1 and SIRT2)to 0.39(SIRT1 and SIRT3).The DAVID platform was also used to perform GO enrichment and KEGG pathway analysis on SIRTs and its 20 interacting proteins.The main cellular components of the above-mentioned interacting protein genes were the nucleus and cytoplasm.They were mainly involved in regulating biological processes such as RNA polymerase ? promoter and DNA template transcription.Their main molecular functions were predicted to be combined with DNA,chromatin and transcription factors.KEGG pathway analysis found that in ovarian cancer,the above genes were mainly involved in important pathways related to tumorigenesis and development such as Notch and FOXO5.A search of the GEPIA database found that the mRNA levels of SIRT5 in ovarian cancer were higher compared to normal tissues,while the mRNA levels of other SIRT members were lower.Using the immunostaining of ovarian cancer tissue microarray,it was found that the protein expression level of SIRT5 in ovarian cancer tissue was significantly higher than that of normal ovarian tissue.In clinical pathological correlation analysis,the expression level of SIRT5 protein was positively correlated with FIGO stage(P<0.0001)and lymph node metastasis(P=0.0019)and negatively correlated with a low grade of differentiation(P=0.0179).Online Kaplan-Meier analysis suggests that high expression of SIRT5 is associated with poor chemotherapy response in patients with ovarian cancer(P=0.0018)6.Cell immunofluorescent experiments found that SIRT5 is mainly located in the cytoplasm and a small amount in the cell nucleus.Among the three ovarian cancer cell lines,A2780 is cisplatin-sensitive,SKOV-3 and CAOV-3 are cisplatin-resistant cells,and the IC50 values are 9.362±0.489?g/ml,39.743±4.756?g/ml,and 80.813±7.058 ?g/ml,respectively.The WB showed that SIRT5 expression was higher in SKOV-3 and CAOV-3 cisplatin-resistant cells than in A2780 cisplatin-sensitive cells.DDP can raise the expression level of SIRT5 in a concentration-dependent manner.7.CCK-8 and colony formation experiments found that,compared with the control group,up-regulate SIRT5 in A2780 cells can promote the proliferation of ovarian cancer cell line and improve the cisplatin IC50 value while down-regulate SIRT5 in SKOV-3 and CAOV-3 cells can inhibit the proliferation ability of ovarian cancer cells and reduce their cisplatin IC50 value8.Through WB and cell immunofluorescent experiments,it was found that in the cisplatin-treated ovarian cancer cell line,up-regulate SIRT5 can inhibit the DNA damage caused by DDp,i.e.the level of gamma-H2AX protein decreased and the number of foci formed decreased,while down-regulate SIRT5 got the opposite results9.Through the ROS test kit found that DDP can promote the production of ROS in time-,concentration-dependent manner,and the ROS level reaches a peak at 24 hours of the action of DDP.Up-regulate SIRT5 can inhibit the production of DDP-induced ROS,and the downward adjustment results to the contrary.After using the ROS inhibitor(NAC)and upregulate SIRT5,the inhibition of the protein level and fluorescence foci of y-H2AX were reduced.Meanwhile,SIRT5 was found to be able to positively regulate the expression of the DNA damage repair gene BRCA1,leading to reduce the protein level of ?-H2AX.10.Nrf2 and SIRT5 have a positive correlation,and WB verified that after upregulating SIRT5,Nrf2 and its target gene HO-1 protein expression levels increased while after downregulating SIRT5,resulting in the opposite results.Cell immunofluorescent and cytosolic and nuclear isolation have shown that SIRT5 can promote the nuclear translocation of Nrf2.After 48 hours of administration of Nrf2 signaling pathway inhibitor ML385,Nrf2 expression levels were significantly inhibited,and the inhibition of ROS level induced by SIRT5 was significantly reducedConclusion:1.SIRT1-4,SIRT6 and SIRT7 may be potential biomarkers for the assessment of individualized prognosis in patients with ovarian cancer2.SIRT5 is a potential target for accurate therapy for patients with ovarian cancer3.SIRT5 expression is increased in ovarian cancer tissues and high SIRT5 levels predict poor chemotherapy response4.SIRT5 promotes cell proliferation and cisplatin resistance in ovarian cancer cells5.SIRT5 promotes cisplatin resistance in ovarian cancer by suppressing DNA damage in a ROS-Dependent Manner via regulation of the Nrf2/HO-1 Pathway.