Curcumin Inhibits The Growth And Induces Apoptosis Of Adrenocortical Carcinoma:Identification Of Potential Candidate Genes And Pathways By Transcriptome Analysis

BackgroundAdrenocortical carcinoma（ACC）is an endocrine tumour with high malignancy,invasiveness,and recurrence rates and has a poor prognosis.Most patients are diagnosed at an advanced stage with invasion of adjacent organs or metastatic disease.Currently,complete resection is the only feasible method to cure ACC.However,the prognosis of patients with relapsed or metastatic disease is unfavourable.Chemotherapy remains an effective method of adjuvant therapy in patients at high risk of relapse and metastasis,and mitotane is the only drug approved for the systemic treatment of ACC.However,mitotane has a narrow therapeutic range,poor bioavailability and side effects.Therefore,safe treatment strategies to overcome drug tolerance and minimize side effects are needed to improve ACC treatment.Curcumin is a plant polyphenolic compound and a major component in turmeric（Curcuma longa）.Curcumin has been reported to have good efficacy and biological safety for treating cancer,with targeted inhibitory effects on gastric,colorectal,breast and cervical cancers.In addition,clinical trials have indicated that curcumin is safe and exhibits therapeutic efficacy in patients with progressive,advanced cancers.However,whether curcumin can induce ACC cell apoptosis or inhibit tumour growth is unclear,and the possible mechanisms are undefined.ObjectiveIn the current study,we evaluated the effect of curcumin on ACC and analysed the underlying mechanisms by high-throughput sequencing.Our systemic analysis of curcumin-induced whole-transcriptome alterations provided precise molecular targets and signalling pathways,contributing to the improved application of curcumin for ACC treatment.Methods（1）The effects of curcumin on the proliferation,migration,invasion and apoptosis of ACC cells:SW-13 and NCI-H295R cells were treated with 0,10,20,30,40,50,60,80 or 100μM curcumin for 24 or 48 h.Cell viability was then measured with a Cell Counting Kit-8（CCK-8）assay.In the cell motility assay,both the migration and invasion of SW-13 and NCI-H295R cells were treated with 0,20,30 or 40μM curcumin for 24 h.Then,we performed Hoechst 33258staining and flow cytometric analysis to investigate the effects of curcumin on SW-13 and H295R cell apoptosis.（2）RNA library preparation,high-throughput sequencing and mRNA-seq data analysis:total RNA was extracted from control-and curcumin-treated groups of SW-13 cells.PCR amplification,product purification and quantification,and sequencing were carried out with the Illumina Hi Seq 2500platform.Fast QC（version 0.11.2）was used for evaluating the quality of sequenced data.BEDTools（version 2.26.0）was used to statistical analysis the gene coverage ratio and the distribution of sequenced sequence on chromosome.The significant differentially expressed genes（DEGs）were obtained using a threshold of q-value<0.05 and a fold change>2.Top GO（version 2.24.0）was used for Gene Ontology（GO）enrichment.Cluster Profiler（version 3.0.5）was used for Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis.Protein-protein interaction（PPI）network was performed with R igraph package.Real-time fluorescent quantitative PCR（RT-qPCR）and Western blot analyzed the potential candidate genes and pathways.（3）Short hairpin RNA（sh RNA）lentivirus transfection:knockdown CHOP and negative control（NC）lentivirus were designed and synthesised.SW-13cells were plated in 12-well plates at a density of 1x10⁵cells/well and incubated overnight.Subsequently,the medium was replaced with medium containing lentivirus（CHOP sh RNA or NC vector）.After 12 h,the medium was substituted with DMEM supplemented with 10%FBS.The expression of green fluorescent protein was observed using fluorescence microscopy after 72 h.The medium was changed by 2μg/m L puromycin complete medium and cells were cultured for 24 h at 37℃ to kill the non-transfected cells.Finally,the medium was exchanged with complete medium.RT-qPCR and western blotting analysis were performed to determine the efficiency of CHOP knockdown expression.Hoechst33258 staining and flow cytometric analyzed the effects of curcumin on SW-13cell apoptosis.（4）Xenograft transplantation and therapy:four-week-old BALB/c nude male mice（18-20 g）were obtained and raised under standard conditions in the Experimental Animal Center of Guangxi Medical University.All animal experiments were approved by the Animal Ethics and Welfare Committee of Guangxi Medical University.SW-13 cells（2×10⁶cells in 100μl of PBS per mouse）were injected subcutaneously into the right flank.The tumours were measured every other day,and the tumour volumes were calculated with the following formula:volume=length×width²×1/2.When the tumour volumes were 100 mm³,the mice were randomly divided into three groups of 8 mice per group.Curcumin dissolved in olive oil（50 or 100 mg/kg）was administered（via intraperitoneal injection）to mice once daily for 2 weeks,while mice in the control group received injections of olive oil.At the end of treatment,the mice were anaesthetized with sodium pentobarbital and then sacrificed using cervical dislocation,and the tumours were removed and weighed for subsequent studies.Electron microscopy observed changes in organelles,and RT-qPCR and Western blot analyzed the expression levels of the related genes in SW-13 xenograft tumours.Results（1）Curcumin inhibits the viability,migration and invasion of ACC cells:the CCK-8 assay was used to evaluate the viability of SW-13 and NCI-H295R cells.We exposed SW-13 and NCI-H295R cells to various concentrations of curcumin（0,10,20,30,40,50,60,80 or 100μM）for 24 h or 48 h.Cell viability decreased significantly in a concentration-dependent manner.We performed a dose-response study to determine the half-maximal inhibitory concentration(IC₅₀)values for curcumin.The IC₅₀values for curcumin in SW-13 and NCI-H295R cells were 50.809±1.706 and 59.271±1.773 at 24 h and 35.917±1.555 and 42.317±1.627 at 48 h,respectively.In the cell motility assay,both the migration and invasion of SW-13 and NCI-H295R cells were suppressed by exposure to curcumin for 24 h.Compared with the control cells,SW-13 and NCI-H295R cells treated with 40μM curcumin exhibited markedly reduced cell migration and invasion abilities（P<0.01）.（2）Curcumin induces the apoptosis of ACC cells:to investigate the effects of curcumin on SW-13 and H295R cell apoptosis,we first examined the cell morphology during the response to the treatment.Compared to the control groups,the groups treated with curcumin for 24 h and subjected to nuclear staining with Hoechst 33258 contained an increased number of cells with a reduced nuclear size,chromatin condensation,nuclear fragmentation,the appearance of apoptotic bodies,all of which are characteristics of apoptotic cells.Next,we performed flow cytometric analysis with Annexin V-APC/7-AAD and found that curcumin induced apoptosis in a dose-dependent manner（P<0.01）.（3）RNA-seq and bioinformatic analyses of the mechanism of curcumin in ACC:we determined the experimental conditions for transcriptome analysis based on cell viability assays.CCK8 assay results showed that 50μM curcumin,which resulted in a cell viability of 49.9%,was the closest to the IC₅₀of curcumin in SW-13 cells after incubation for 24 h.Therefore,transcriptome analysis was performed on SW-13 cells after treatment with 50μM curcumin for24 h.In total,385 DEGs were identified in the curcumin-treated group compared with control group;114 genes were upregulated,and 271 genes were downregulated.GO enrichment analysis was used to examine the processes in which the DEGs were involved,and the top ten enriched GO terms were associated with in cell cycle,cell proliferation,cell migration,cell differentiation and execution phase of apoptosis.KEGG enrichment and PPI analyses,the curcumin-induced apoptosis of ACC cells is mediated primarily through the cell cycle,micro RNAs in cancer,ER stress and MAPK signalling pathways.These altered genes and pathways may enhance our understanding of the mechanisms underlying the curcumin-induced apoptosis of ACC cells.We validated these results by RT-qPCR and Western blot analysis.HSP70 expression was downregulated,which may have increased JNK and p38 phosphorylation,after curcumin intervention（P<0.05）.Activation of JNK and p38 increased the binding of downstream c-Jun and c-Fos and promoted the expression of the apoptotic precursor factor ATF4,which led to upregulation of the ER stress-associated apoptotic factor CHOP to induce ACC cell apoptosis（P<0.05）.Moreover,expression of the apoptotic factor Bax increased,while expression of the anti-apoptotic factor Bcl-2 decreased（P<0.05）.These results showed that the JNK,p38 MAPK and ER stress pathways were activated.（4）Knockdown of CHOP expression inhibits curcumin-induced SW-13cell apoptosis:to further determine that CHOP plays an important role in curcumin-induced apoptosis,sh RNA was used to knock down CHOP expression.CHOP mRNA expression was reduced by approximately 70%compared with that in cells transfected with the NC group.Following treatment with 40μM curcumin,there was a significant reduction in CHOP expression compared with the curcumin-treated group（P<0.01）.Moreover,the protein expression of the apoptotic factor Bax decreased,while the protein expression of the antiapoptotic factor Bcl-2 increased（P<0.01）.Knockdown of CHOP expression inhibited curcumin-induced SW-13 cell apoptosis（P<0.01）.（5）Curcumin inhibits SW-13 xenograft tumour growth:based on the inhibitory effects of curcumin on ACC cell growth in vitro,we established SW-13 xenograft tumour models in nude mice.After intraperitoneal injection of50 or 100 mg/kg curcumin or vehicle alone for 2 weeks,we observed a significant reduction in the tumour weight and volume in the groups treated with50 and 100 mg/kg curcumin（P<0.05）.The body weights of the mice in the different treatment groups did not change significantly（P>0.05）.Curcumin at a dose of 100 mg/kg showed a greater inhibitory effect on tumour growth than either of the other treatments,and expansion of the ER was observed under an electron microscope.The levels of the related genes in the JNK,p38 MAPK and ER stress pathways were measured in xenograft tumours by RT-PCR and Western blot analysis.After curcumin intervention,HSP70 and Bcl-2 expression levels were decreased,while Bax expression levels were increased（P<0.05）.The JNK,p38 MAPK and ER stress pathways were activated,and the levels of p-JNK,p-p38,c-Jun,c-Fos,ATF4,and CHOP were significantly increased in the 50 mg/kg and 100 mg/kg curcumin-treated groups（P<0.05）.Conclusion（1）Curcumin inhibits the viability,migration,invasion and induces the apoptosis of ACC cells.（2）Curcumin activates JNK and p38 MAPK and stimulates ER stress,which plays important roles in apoptosis.（3）CHOP is a potential functional target gene in curcumin-induced ACC cell apoptosis.Knockdown of CHOP expression inhibits curcumin-induced SW-13 cell apoptosis.（4）Curcumin is well tolerated and effectively inhibits tumour growth in vivo in a nude mouse xenograft model.JNK,p38 MAPK and ER stress pathways are the predominant pathways associated with curcumin-inhibited SW-13 xenograft tumour growth.