The Study Of Curcumol On Proliferation And Apoptosis Of Multiple Myeloma Cells

BackgroundMultiple myeloma（MM）,a relatively common hematologic malignancy,originates in neoplastic plasma cell disorder.It often occurs in middle-aged and elderly patients and has a higher incidence.At present,the incidence of MM in China is about 1/100000 to 2/100000,which has exceeded acute leukemia and ranks second in the incidence of hematological malignancies.In recent years,with the intensification of the aging population in China,the incidence of the disease has gradually increased.The main pathological characteristic is the abnormal proliferation of monoclonal plasma cells in bone marrow.The clinical manifestations mainly include osteolytic damage,anemia,hyperviscosity,renal function damage and repeated infection.Myeloma bone disease（MBD）is one of the most common complications,resulting in diffuse osteoporosis and localized destruction,severely affecting the life quality and prognosis of multiple myeloma patients.Recently,MBD has been recognized to be the result of metabolic imbalance.The interaction of cytokines secreted by multiple cells like multiple myeloma cells,osteoclast and osteoblast existing in the microenvironment results in the hyperactive of osteoblasts and inactive of osteoclasts,causing myeloma bone disease.At present,the treatment of MM mainly includes traditional routine chemotherapy and hematopoietic stem cell transplantation.Although allogeneic hematopoietic stem cell transplantation can cure some patients,high mortality rate limits the clinical use.Conventional chemotherapy and autologous stem cell transplantation can give patients temporary relief,but the high recurrence rate and the emergence of multidrug resistance make it difficult to treat multiple myeloma.In the past few years,the application of newly medicines like proteasome inhibitors has improved the prognosis of multiple myeloma,but it is still considered as an incurable disease.Thus,seeking for the potential mechanism of multiple myeloma induced osteocyte death and drugs or treatments to improve the prognosis of patients is of important significance.Curcumol（Cur）,also known as turmeric curcuma alcohol,is one of the important components of the volatile oil in curcuma zedoary（zedoary,dried root rhizome and Curcuma wenyujin Guangxi Lotus）.Zedoary turmeric oil contains 68%-70%Curcumol,which plays an important part in the effects of anticancer,antibacterial,antiviral.The traditional Chinese medicine holds the theory that the curcuma zedoary is acrid-bitter flavored and warm in nature,with the effects of invigorating the circulation of blood and relieving pain.Modern pharmacological studies have shown that zedoary alcohol has the anti-hepatoma and hepatoprotective effects with the advantages of low toxicity and no reduction in the number of white blood cells.Previous studies in vitro have shown that zedoary alcohol can effectively inhibit the proliferation of ovarian cancer,cholangiocarcinoma,kidney cancer,prostate cancer and liver cancer.The main anti-tumor mechanism is induction of apoptosis and cycle arrest.However,the effect of zedoary alcohol on human multiple myeloma is still unknown.In this study,we will screen the differentially expressed genes（DEGs）through analysis of gene expression profile to uncover the potential mechanism of multiple myeloma induced osteosyte death and to investigate the anti-tumor effect of Curcumol on multiple myeloma and the underling mechanism.PartⅠIdentification of candidate genes for multiple myeloma-induced osteocyte death based on microarray dataObjective:1.To observe the differentially expressed genes and related signaling pathways by analyzing the microarray.2.To construct a protein-protein interaction（PPI）network to further explore the molecular mechanism of bone cell death in MM patients based on the microarray.Methods:1.Affymetrix microarray data:Gene transcriptional profile of GSE27372[10]was downloaded from Gene Expression Omnibus（GEO）database（https://www.ncbi.nlm.nih.gov/geo/）,and GSE27372 was based on the platform of GPL570[HG-U133_Plus₂]Affymetrix Human Genome U133 plus 2.0 array.There were six specimens in this dataset,including three HOB-01（osteocyte cell line）control samples and three HOB-01 samples co-cultured with JJN3（human myeloma cell line）.2.DEGs screening:AFFY package（version 1.28.0,http://www.bioconductor.org/packages/release/bioc/html/affy.html）of Bioconductor was used to pre-process the Affymetrix microarray data.All raw data were scaled according to the robust multi-array average（RMA）method with default settings.After background correction,quantile normalization,and probe summarization,the gene expression matrix was obtained.The Student’s t test method was adopted to analyze the expression differences between control and co-culture group.For each significant DEG,both P value<0.05 and log2 fold change（FC）>0.58 need to be met.3.Function and pathway enrichment analyses of DEGs:Functional annotation for DEGs was performed by Gene Ontology.Kyoto Encyclopedia of Genes and Genomes（KEGG http://www.genome.jp/kegg/pathway.html）pathway enrichment analysis was used to identify main functional and metabolic pathways involving DEGs.We used P value<0.01 as the cut-off criterion for the enrichment analysis which was conducted by the Database for Annotation,Visualization and Integrated Discovery（DAVID;version 2.1b,http://david.abcc.ncifcrf.gov/）online software.4.Transcriptional factors（TFs）and tumor-associated genes（TAGs）in DEGs with the function of transcriptional regulation were selected based on the TRANSFAC database（http://www.gene-regulation.com/pub/databases.html）.According to the TSGene database（http://bioinfo.mc.vanderbilt.edu/TSGene/search.cgi）and TAG database（http://www.binfo.ncku.edu.tw/TAG/）,we extracted the TAGs from DEGs,including tumor suppressor genes and oncogenes.5.Construction of PPI network and sub-network.The interactional pairs of DEGs were analyzed via online tool Search Tool for the Retrieval of Interacting Genes（STRING;version 9.0,http://string-db.org）,and the datas were downloaded on June 27,2014.Only the gene pairs which were recorded in database,and experimental validated,text-mined,or co-expressed were used and combined score>0.4 were set as the criterion of PPI.Then,the PPI network was constructed using Cytoscape（version 2.8,http://cytoscape.org）.The sub-network analysis of PPI network was performed using the ClusterONE plug of Cytoscape.The pathway enrichment analysis of the genes in the most significant sub-network was performed by using the DAVID online software.Results:1.Identification of differently expressed genes:After analyzing the microarray data of control and coculture groups,a total of 393 DEGs were screened out,including 167 down-regulated genes and 226 upregulated genes in co-culture group.2.Signaling pathways and biological functions associated with DEGs:To explore the specific functions and pathways of DEGs,functional and pathway enrichment analyses were performed.The down-regulated genes were most significantly enriched in the functions of cardiovascular system development,circulatory system development,and the pathways of protein digestion,absorption,and extracellular matrix（ECM）-receptor interaction pathway.Especially,epidermal growth factor（EGF）,sphingosine-1-phosphate receptor 1（S1PR1）and Neuropeptide Y1 receptor（NPY1R）were involved in circulatory system development.Moreover,the upregulated genes were most significantly enriched in the functions of regulation of response to stimulus and regulation of signaling function,as well as the pathways of JAK-STAT signaling,nicotinate and nicotinamide metabolism.3.Transcriptional factors and tumor associated genes:The TFs and TAGs differentially expressed between control and co-culture groups were further extracted from DEGs.As shown in Table 3,4 downregulated TFs and 18 up-regulated TFs（e.g.,Kruppel like factors 4,KLF4;and IFN regulatory factor-8,IRF8）were discovered.Furthermore,16 down-regulated TAGs and 21 up-regulated TAGs were screened out（Table 3）.Among the down-regulated TAGs,there were 10 tumor suppressor genes and 3 oncogenes.Meanwhile,among the up-regulated TAGs,there were 10 tumor suppressor genes（e.g.IRF8）and 9 oncogenes.4.Interactions between DEGs:The interactions were identified using STRING software.By integrating DEG pairs with combined score>0.4,a PPI network was built,consisting of 89 down-regulated genes and 113 up-regulated genes.A total of 10 DEGs had connectivity degree larger than 15,e.g.,EGF（degree=31）and early growth response 1（EGR1,degree=19）.Besides,EGF could interact with EGR1 in the PPI network.5.Construction of Sub-network:A sub-network was obtained from PPI network（P=0.00043）,consisting of 9 significant DEGs like S1PR1,complement-3 a receptor 1（C3AR1）and NPY1R.In the sub-network,S1PR1,C3AR1 and NPY1R had interactions with each other.Furthermore,the up-regulated C3AR1,as well as the down-regulated S1PR1 and NPY1R in the sub-network were enriched in the neuroactive ligand-receptor interaction pathway.Conclusions1.The differently expressed genes might be involved in osteocyte cell apoptosis induced by MM cells.2.We studied the potential molecular mechanism of the increased osteocyte death,which may provide a theoretical basis for understanding and treatment of osteolysis in MM patients.Part ⅡAnti-tumour Effects of Curcumol on Multiple Myeloma Cells and the underlying MechanismObjectives:1.To observe the effects of Curcumol on human Multiple myeloma cells.2.To investigate the possible signaling pathway and molecular mechanism of the anti-tumour effects of Curcumol on human multiple myeloma.Methods:1.The human multiple myeloma cells were subjected to different concentrations of Curcumol for 24h,48h,72h and 96h.The cell viability was determined by using CCK-8 assay to analyze the effect of Curcumol on the proliferation of human multiple myeloma cells.2.Flow cytometry and cell apoptosis detection kit were used to detect the effects of different concentrations of Curcumol on the apoptosis rate of human multiple myeloma cells.Western Blot was used to detect the expression of apoptosis related proteins such as Bcl-2,Survivin and Bax to analyze the possible molecular mechanism.3.Flow cytometry and cell cycle assay kit were used to detect the effect of different concentrations of Curcumol on human multiple myeloma cell cycle distribution.The expressions of cell cycle related proteins cyclin D1,p21 and p27 were detected by Western Blot to analyze the possible molecular mechanism of G0/G1 cycle arrest in human multiple myeloma cells.4.Western Blot was used to detect the effect of Curcumol on the TLR4/NF-κB signaling pathway in human multiple myeloma cells and analyze the possible molecular mechanism of the inhibitory effect of Curcumol on human myeloma cells.5.Based on our previous microarray data analysis,we used RT-PCR to validate some of the key genes in our cell model.Results:1.The effect of curcumol on the proliferation of human multiple myeloma cells:After treatment with different concerntrations of curcumol for 24h,48h,72h and 96h,CCK-8 assay was used to detect the proliferation of human multiple myeloma cells.The results showed that curcumol could significantly inhibit the cell proliferation of human multiple myeloma cells in a dose-and time-dependent manner.2.The effect of curcumol on the apoptosis of human multiple myeloma cells:Flow cytometry and cell apoptosis detection kit were used to detect the effect of curcumol on the apoptosis rate of human multiple myeloma cells.The results showed that curcumol could significantly induce apoptosis of tumor cells compared with the control group.And with the increase of the concentration,the apoptosis of the cells was obviously increased.Next,we examined the protein expression of Bcl-2,Survivin and Bax in myeloma cells treated with curcumol.We found that the anti-apoptotic protein Bcl-2 and Survivin were significantly downregulated after treatment with curcumol for 48h compared with the control group.However,the protein expression of pro-apoptotic protein Bax increased slightly in myeloma cells exposed to curcumol for 48h.3.The effect of curcumol on the cell cycle of human multiple myeloma cells:Flow cytometry and cell cycle assay kit were used to detect the effect of curcumol on human multiple myeloma cell cycle distribution.The results showed that curcumol could cause significant accumulation of cells in G0/G1 phase in a dose-dependent manner.Western Blot results showed that the expression of cyclin D1 was remarkably down-regulated,while p21 and p27 protein were significantly up-regulated.4.The effect of curcumol on TLR4/NFNF-Kb signaling pathway in human multiple myeloma cells:Western Blot was used to detect the influence of curcumol on the TLR4/NF-κB signaling pathway in human multiple myeloma cells.Compared with the control group,the protein expression of TLR4 and p-NF-κB in cells treated with curcumol was obviously increased,indicating that curcumol might induce cell apoptosis and cell cycle arrest and inhibit the proliferation of multiple myeloma cells by activating the TLR4/NF-κB signaling pathway.5.The effect of curcumol on related genes in human multiple myeloma cells:Based on our previous microarray data analysis,we used RT-PCR to validate some of the key genes in our cell model.After exposed to curcumol for 48h,the mRNA expressions of S1PR1 and EGF were markedly downregulated,while the expressions of C3AR1 and EGR were significantly upregulated.The target genes that may play an important role in osteocyte death induced by multiple myeloma were further validated.Conclusions:1.Curcumol could significantly inhibited the proliferation of human multiple myeloma cells in a dose-and time-dependent manner.2.Curcumol could induced apoptosis and G0/G1 phase cell arrest of human multiple myeloma cells via activating of TLR4/NF-κB signaling pathway.Part ⅢAnti-tumor Effects of Curcumol on Multiple Myeloma Cells tumor-bearing miceObjectives:1.To identify the anti-rumor effects of Curcumol on human Multiple myeloma cells in vivo.2.To confirm the underlying molecular mechanism of Curcumol against human multiple myeloma in vivo.Methods:1.To identify the potential anti-tumor effects of Curcumol on human Multiple myeloma cells in vivo.The established human Multiple myeloma cells tumor-bearing mice model were received indicated concentrations of Curcumol for 21 days.During the procedure,the volume of tumor was measured.At the end of experiment,the mice were sacrificed,and tumors were removed to weigh for calculating inhibition rate of tumor growth.2.To confirm the underlying molecular mechanism of Curcumol against human multiple myeloma in vivo.The expressions of Bcl-2,Survivin and Bax were measured by Western Blot.3.To validate whether Curcumol affected TLR4/NF-κB signaling pathway and subsequently induced cell apoptosis in tumor tissues.The expressions of TLR4 and NF-κB were measured by Western Blot.4.To further prove the acquired data in vitro,the expressions of S1PR1,EGF,C3AR1,and EGR were analyzed by quantitative PCR and Western Blot.Results:1.Curcumol inhibits the growth of human multiple myeloma cells tumor tissues:After treatment with different concentrations of Curcumol for 21 days,the volume and weight of tumor tissues were measured,and the inhibition rates of tumor tissues were calculated.The results showed that Curcumol significantly inhibited the growth of human multiple myeloma cells tumor tissues in a dose-dependent manner.2.Curcumol affects the expressions of apoptosis-assocaited proteins in tumor tissues:The expressions of Bcl-2,Survivin and Bax in tumor tissues were measured by Western Blot.The results showed that Curcumol significantly reduced the protein expressions of anti-apoptosis Bcl-2 and Survivin,while elevated the expression of pro-apoptosis Bax when compared with the control group,which were consistent with the results in vitro.3.Curcumol affects TLR4/NF-κB signaling pathway in tumor tissues:The expressions of TLR4 and NF-κB in tumor tissues were measured by Western Blot.The results showed that Curcumol significantly induced the expressions of TLR4 and NF-κB in tumor tissues.4.Curcumol modifies the expressions of bone cells apoptosis-associated genes in tumor tissues:the expressions of bone cells apoptosis-associated genes S1PR1,EGF,C3AR1 and EGR were measured by RT-PCR and Western Blot.The results revealed Curcumol significantly up-regulated the mRNA and protein expressions of C3AR1 and EGR.At the same time,Curcumol markedly down-regulated the mRNA and protein expressions of S1PR1 and EGF compared with control group.Conclusions:1.Curcumol significantly inhibited the growth of human multiple myeloma cells tumor tissues,and the potential mechanism might be attributed to Curcumol-induced activation of TLR4/NF-κB signaling pathway and subsequent cell apoptosis.2.Curcumol affected the expressions of bone cells apoptosis-associated genes including S1PR1,EGF,C3AR1 and EGR,which were involved in tumor tissues-induced bone cells apoptosis in vivo.