Utilizing IL-8Promoter Reporter Gene Assay Screening Of Single Component Which Can Inhibit Secretion Of IL-8in Lung Cancer Microenvironment

Objective:According to the former research, IL-8is closedly related to angiogenesis which canbinfluenc the survival rate of patient with tumor. We search the single compound of natureproduct which have the capacity of inhibit lung cancer associated with cells secretion of IL-8.Try to find herbal ingredients that can make IL-8expression decrease, in turn, can affect thegrowth and metastasis of cancer cells. We do not want to tumor cell death that is we hope tofind an immunomodulatory agent, regulating the microenvironment of the tumor and easingof tumor progress throughout the trial.Method:First, IL-8promoter constructed pGL-3Basic plasmid was transfected into HEK293cells, over4h, natural compounds with IC20concentrations for24hours, followed bymeasuring fluorescence through reporter assay system..Then, taking advantage of MTT assayto detect cytotoxin of different natural single compound in the HEK293cell lines.Afterscreening,We focused on2',4'-dihydroxy-4',6'-dimethyloxy chalcone. Finally, In order tostudy that the secretion of IL-8inflenced by2',4'-dihydroxy-4',6'-dimethyloxy chalcone(chalcone) in lung adenocarcinoma.This study dealt with three types of cells of CL1-0, CL1-5,THP-1respectively and analysis of the compound of the above three types of cells expressionof IL-8. In this study, real-time quantitative PCR, ELISA, reporter gene analysis method todetection of IL-8expression and EMSA was used to analyze the regulatory mechanism.Result:1．We preformed to screen single compounds in HEK293cell line. Finally13kinds ofsingle compounds were comformed that IL-8product decreased after treatment, which mainlyinclude sesamin and2',4'-dihydroxy-4',6'-dimethoxy chalcone. 2. Taking advantage of fluorescent reporter gene assay platform.2',4'-hydroxy-4',6'-dimethoxy-chalcone has a good ability to inhibit IL-8promoter activity in CL1-0cells. Theinhibitory effect was up to50%.3. Real-time PCR method detected IL-8mRNA levels after treating CL1-5cells by2',4'-dihydroxy-4',6'-dimethoxy chalcone. IL-8mRNA levels significantly increased. The effectof enhanced expression increased by30%.4. Carrying EMSA out after2',4'-dihydroxy-4',6'-dimethoxy chalcone processing ofCL1-5cell line. We found that the binding capacity between the binding sites of IL-8upstream promoter and AP-1and NF-κB as the IL-8transcription factor was significantlyimproved.5. Tumor-associated macrophages(THP-1) droped secretion of IL-8after processing2',4'-hydroxy-4',6'-dimethoxy chalcone. Phenomenon of the inhibitory effect of IL-8expressionreached to24.9%.Conclution：1.2',4'-dihydroxy-4',6'-dimethoxy-chalcone for early-stage lung cancer cells andtumor-associated macrophages have a positive effect. This compound can inhibit secretion ofIL-8of the CL1-0and tumor associated macrophages (THP-1). The adjustment ofinflammatory cytokines IL-8releasing through the compound in the early-stage tumor wasobserved. Thereby regulating the microenvironment of tumor cell growth, and achieving thepurpose of alleviating lung carcinoma progress.2.2',4'-dihydroxy-4',6'-dimethoxy chalcone for lung adenocarcinoma tumor cells withhigher levels of worse has a negative effect such as CL1-5. This compound can increaseCL1-5cells to secrete IL-8. In Contrary, Tumor-associated macrophages to the tumor itselfcan be influenced by autocrine and paracrine that can not be overlooked, it is notrecommended for patients with advanced lung cancer tumor cells to administrat thiscompound.