Study On Radiosensitization Effect Of Curcumin On Human Nasopharyngeal Carcinoma Cell

Background and ObjectiveNasopharyngeal carcinoma(NPC),which accounted for2.81%of national malignant tumor mortality, is one of the most common malignant tumors in China.The morbidity of NPC in China accounted for more than80%of the worldwide according to WHO estimated.The incidence of NPC was higher in Guangdong, Guangxi, Hunan and other provinces of southern China,especially in Guangdong Pearl River Delta region and XiJiang basin was highest. The incidence was15-30/10million population, the mortality was18.46/10million population.The NPC patients were treated mainly by radiotherapy, the average of5-year survival rate was50%-60%after radiotherapy.However,the early symptoms of nasopharyngeal carcinoma was not obvious,70%patients with advanced stage when diagnosed, the5-year survival rate of advanced nasopharyngeal carcinoma (Ⅲ,Ⅳ) is lower than those in primary cases, which nearly10%-40%.The main causes of treatment failure were distant metastasis and local recurrence.As a dose-dependent tumor, the local control rate and survival rate of nasopharyngeal carcinoma could increase by increasing local radiation dose in the course of medical treatment.However, patients can occur radiation reaction after radiotherapy, especially acute radiation reaction, significantly affect the patient’s quality of life and the processes of radiotherapy, cause tremendous damage and suffering to the patient.Therefore,it is important to find an effective radiosensitizers prevent side effects of irradiation.5-fluorouracil and cisplatin were commonly used radiosensitizers at present.They kill cancer cells, meanwhile, has a toxic effect on normal tissue cells, which limits their application in clinical. In order to improve overall survival in nasopharyngeal carcinoma patients,looking for a safe and low-toxicity radiosensitizing drugs and demonstrate its mechanism of radiosensi tization have important, clinical value and scientific sense.Curcumin (Cur) is a effective polyphenol which is extracted from plant of Zingiberaceae and Curcuma genus. A large number of preclinical cell research, animal experiment and phase II clinical study indicated that Curcumin possesses widely pharmacological activities:anti-proliferation,anti-angiogenesis and resistance to invasion.It could regulate resistance to drugs and radioresistance of the body, promote wound healing,and has excellence efficacy in diabetes,Alzheimer’s disease, Parkinson’s disease,cardiovascular disease,lung disease and joints disease.In recent years studies, we founded that Curcumin significantly inhibit culture, proliferation, metaptosis of various tumor cells,such as colon cancer, breast cancer, liver cancer, ovarian cancer and others.Previous studies showed that Curcumin not only inhibit proliferation and induce apoptosis in tumor cells,but also has radio sensitization effect on tumor.The radiosensitization effect of Curcumin on nasopharyngeal carcinoma and its mechanism was not clear.Therefore,we studied the radiosensitization effect of Curcumin on nasopharyngeal carcinoma and its mechanism.Research at home and abroad confirmed that multidrug resistance gene1(MDR1) is closely related to radiosensitivity.SongPeng confirmed that the expression of MDR1was increased after irradiation on tumor cells in vitro,and have tolerant to radiotherapy subsequently,which indicated that overexpression of MDR1causes the cell obtain radioresistance.The expression of MDR1is regulated by various factors,but microRNA targeted regulation effect for MDR1has not reported yet. In our prophase research, we confirmed that MicroRNA regulates expression of MDR1. Therefore,we confirmed radiosensitization effect of Curcumin on nasophar yngeal carcinoma in vivo and in vitro.We observe the expression of MDR1proteins and the explore microRNA targeted regulation of MDR1,consider the possibilities of microRNA involve in radiosensitivity of nasopharyngeal carcinoma.Our study provide new ideas for reasonable protection on normal tissue in the radiotherapy process.Long non-coding RNA (IncRNA) refers to the transcript length more than200NT (nucleotide) and is not translated into protein functional RNA molecules.lncRNA can affect expression of target proteins that encoded by specific locus, regulatory activity of protein-binding factor, guiding position of chromatin modifying compound,and produces a large number of small RNA with5’ Cap structure after transcriptional processing. In recent years, studies have shown that IncRNA is closely related with occurrence and development of many cancers such as bladder cancer, breast cancer, liver cancer, and prostate cancer. In earlier research, we found that pretreated with Curcumin,part of lncRNA’s expression were changed. Therefore, whether lncRNA participated in nasopharyngeal carcinoma and How lncRNA play the role,Which were one of our research aim.Contents and methods1.The antiproliferative effect of Cur by MTT assayHuman nasopharyngeal carcinoma CNE-2cell used in this study, The cytotoxic effects of Curcumin on CNE-2cell detected by MTT. CNE-2cell were treated with Cur at increasing concentrations (5-40μM) for24and48h,and a solvent control group were setted.The absorbance was recorded at490nm by microplate reader. The IC50was calculated by survival fractions2. The clone formation experimentsExperiments were divided into irradiation and irradiation combined with Curcumin group. Cells were trypsinized,diluted,counted,and seeded into six well cell culture plate at densities of100-3,000cells/well. Thereafter, cells were either left untreated or treated with Cur(10μM) and/or radiation (0-8Gy). After incubating for14days, cells were fixed with methanol and stained with Giemsa.Cell colonies that contained at least50cells were manually counted under adissecting microscope.The cell survival curve was fitted according to single-hit multi-target model and linear-qua dratic model.The parameters of radiosensitivity Do, Dq, α, β was calculated to compare radiosensitization effect of Curcumin on on CNE-2cell.3. Cell cycle was analysised by FCM Experiments are divided into4groups:control group, irradiation group, Curcumin group, irradiation+Curcumin Group (10μM).Culture24h after receiving treatment, and then PI stained. Each phase change of cell cycle was detected by flow cytometry confirmed confirm whether curcumin or radiation (IR) will cause the CNE-2cell block in G2phase.4.The establishment of human nasopharyngeal carcinoma CNE-2nude mice transplant modelThe model was made by cell suspension. The male nude mice were randomly divided into5groups acoording to tumor volume after model was successful,6in every group:control group (saline) group,Curcumin group,irradiation group and Curcumin combined with irradiation group.The Combined treatment group were divided into2subgroup according to drug dosage.We observe the tumor growth and draw tumor growth curve of transplant tumor5.The expression of MDR1and miR-593detected by qPCRExtracted total RNA from CNE-2cell and tumor tissues, reverse transcription into cDNA and PCR amplification with primers.2-△△Ct value represents the relative gene expression strength.6.The expression of MDR1analysised by Western blotWestern blot analysis was performed according to the standard method with antibodies to MDR1and actin after total protein were extracted and quantificated by BCA method7.The dual-luciferase assay determined by dual luciferase reporter gene assay kit.Before transfection,293FT cells (2×105) were seeded into24-well plates, added500μl medium without antibiotics into each well.Transfection was carried after growth of cell fusion reached95%, collected filtered cell and detected dual-luciferase activity after transfected48h.Relative luciferase activity was normalized with Renilla luciferase activityand then compared wim the pGL3control.8. lncRNA microarry assayPick out lncRNAs that express more than2times fold changing by gene chip screening,6differential expression lncRNA such as AK294004was picked out, real-time quantitative PCR technique verified the results.9. Regulative relationship between AK294004and CCND1Target genes of AK294004was predicted by bioinformatics technology. Overexpression, siRNA and AK294004dual-luciferase report system validated binding sites of AK294004and CCND1.Results1. Different concentrations of Curcumin on human nasopharyngeal carcinoma CNE-2cell, both showed a concentration-dependent in24h and48h,inhibitor concentration50%(IC50) of24h and48h were:32.006μmol/1,11.854μmol/1.2. The results of clone formation assay showed that Curcumin reduces colony forming efficiency of CNE-2cell after exposure.The survival curve was fitted according to survival fraction and the indicators that represent radiosensitivity was calculated, which showed that Curcumin enhanced radiosensitivity.Radiation enhancement ratio (SER) was1.33. according to single-hit multi-target model.3. The cell cycle was determined by flow cytometry,the results showed that irradiation lead to CNE-2cell G2/M arrest (P<0.05). After treating with10μM Cur for24h,G1phase significantly reduced (P<0.05), G2phase increased slightly, when IR combined with Cur,compared with the control group, the G2phase significant increased,S phase significantly reduced (p<0.05).4. The tumor weight in each group was (0.83±0.17)g、(0.75±0.23)g、(0.55±0.27)g、(0.47±0.11)g,(0.53±0.23)g respectively.Difference between groups has statistical significance (P<0.05). The volume of xenograft was increasing with time change, the growth rate of the control group was the largest.Compared with the control group,the tumor inhibition rate of Curcumin group, irradiation groups and combination group was33.73%,9.63%and43.37%respectively, Which has a statistically significant difference (P<0.05).5. The results of western bolt showed that the expression of MDRl in the irradiation group significantly up-regulated(P<0.05),while the expression of MDRl in Curcumin (low) combined with irradiation group down-regulated significantly(P<0.05).6. The results of fluorescent quantitative PCR showed that both in cell and tissue,the expression of MDR1in the irradiation group significantlyup-regulated, while the expression of MDR1in Curcumin combined with irradiation group down-regulated significantly((t=2.918,P=0.019; t=2.682;P=0.028).In contrast, the expression of miR-593in the irradiation group significantly down-regulated,while the expression of miR-593in Curcumin combined with irradiation group up-regulated significantly(t=2.918,P=0.019;t=-2.863,P=0.021).7. pGL3-MDRl3’UTR and miR-593were cotransfected, the activity of luciferase was significantly decrease(P<0.01);while pGL3-MDRl3’UTR and miR-593inhibitor were cotransfected, the activity of luciferase was significantly increased(P<0.07). Above all,MDR1and miR-5933’UTR were specific binded.8. Picked out2480coding protein genes.2795lncRNA gene, Which was obvious upregulaed or downregulated after radiation and irradiation combined with Curcumin in CNE-2. real-time quantitative RT-PCR confirmed differentially expressed of AK294004and others wre consistent with microarray results.9. In the overexpression experiments, compared with control cells (pCDNA3-NC), AK204004expression was increased3.16-fold(t=-13.477,P<0.001), while CCND1expression was decreased by46%(t=-12.499,P<0.001) in pCDNA3-AK cells,. In the RNA interference experiments, when compared with control cells (siRNA-NC), AK294004expression was knocked down67%by siRNA-2, and75%by siRNA-1, while CCND1expression was increased1.34and1.48-fold, respectively.A luciferase-based reporter was constructed to evaluate the effect of AK294004direct binding to the putative target sites on the3’UTR of CCND1, for pGL3-W or pGL3-E1construct, pcDNA3-AK significantly lowered luciferase activity compared with pcDNA3-NC(t=14.977,p<0.001; t=4.978,p=0.033). There was no different luciferase activity observed between the pGL3-vector and pGL3-E2constructs(t=3.024, P=0.155; t=1.844, p=0.139).These findings support the hypothesis that IncRNA AK294004directly targets CCND1expression by its exon1 Conclusion1. We confirmed that the radiosensitization effect of Curcumin on nasopharyngeal carcinoma in vitro and in vivo formed by clone formationassay and CNE-2in human nasopharyngeal carcinoma xenograft in nude mice model,2. Western blotting,RT-PCR validated that Curcumin reverse expression of MDR1,miR-593.Dual-luciferase report system make clear that miR-593targeted MDR13’UTR. We confirmed that Curcumin regulated593targetly reverse expression of MDRl that achieve radiosensitization.3. AK294004and other lncRNAs that differential expressed was screened out by gene microarray.Dual-luciferase report system and siRNA make clear that AK294004targeting CCND1.We confirmed that Curcumin regulates AK294004targetly reverse expression of CCNDlto achieve radiosensitization.