The Establishment Of The Technical Platform Based On High-throughput Screening For Tumor Preventive Agents And It's Application In TCM

"The application of natural or synthetic compounds to block, reverse or prevent the occurrence of invasive tumors", as known as chemoprevention, is an effective strategy. Among the diverse carcinogenic factors, the International Agency for Research on Cancer indicated that 80-90% of the development of human tumors are caused by chemical carcinogens. Chemical carcinogens can damage cell DNA to result in gene mutation, and then eventually lead to tumor development. One of the research objectives for chemoprevention is to discover effective drugs with low toxicity. Due to the toxic side effects of artificial chemical drugs, the importance of natural treatment and natural medicine in cancer prevention has gained more and more attention. China has valuable resources for traditional medicine. It has become an urgent need to investigate how to perform high-throughput screening for active preventive ingredients in Chinese herbal medicine.World widely, drug screening models can be generally divided into three types: animal model, tissue and organ model, and cellular and molecular model. The first two models tend to consume a large amount of samples and to use a large number of laboratory animals. They are labour intensive and time consuming. Also, the number of samples being screened each time can be limited and these two models are not suitable for screening for complex active ingredients in Chinese medicine. The drug screening model at the cellular and molecular level has several properties: less sample consumption, high efficiency, understandable drug mechanism, and large sample size screening. However, there is limited research with regard to chemopreventive drug screening at the cellular and molecular level. To our knowledge, we are the first group to report on high-thoughput screening for chemopreventive Chinese medicine through transgenic cell model.This paper aims to establish a simple, highly sensitive and efficient tumor preventive agents screening model to screen for active ingredients in traditional Chinese medicine for cancer prevention.1. The TK promoter was amplified from plasmid pRL-TK by recombinant PCR and the Sac I enzyme restriction site was created. The TK promoter segments were cloned into pMD18-T vector to construct pMD-TK. The plasmids of pMD-TK and pEGFP were double digested with Sal I and BamH I. The TK promoter was connected to the correspondent restriction sites of the vector pEGFP to yield the vector pTK-GFP. The plasmid pTK-GFP was used as a template to amplify TK segments by PCR. The amplified TK segments and vector pEGFP-N1 were digested using Vsp I and BamH I. The TK promoter was ligated to the vector pEGFP-N1 to construct the eukaryotic reporter vector of pTK-GFP/neo. Four synthetic ARE sequences were inserted into the vector pTK-GFP/neo after annealing phosphorylation to regulate the expression of GFP. So the four eukaryotic vectors, pARE-TK-GFP/neo, p2ARE-TK-GFP/neo, p3ARE-TK-GFP / neo and p4ARE-TK- GFP/neo, were constructed respectively first in China.2. The eukaryotic reporter vectors pTK-GFP/neo, pARE-TK-GFP/neo, p2ARE-TK- GFP/neo and p4ARE-TK-GFP/neo were transfected into HepG2 cells respectively. After 24 hours of incubation, the green fluorescence was obvious under the fluorescent microscope for the four types of cells, indicating that the choice of promoter was appropriate. First time in China, the clones resistant to 600μg/ml G418 were isolated to construct the ARE free cell model of HepG2-TK-GFP for the negative control and different ARE repeated sequence control cell models of HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP, HepG2-3ARE-TK-GFP and HepG2-4ARE-TK-GFP.3. After HepG2-TK-GFP, HepG2-ARE-TK-GFP, HepG2-2ARE-TK-GFP and HepG2- 4ARE-TK-GFP were amplified, they were digested by trypsin and seeded into wells of a black 96-well plate with 5×104 cells in each well. After the incubation for 24 hours, in addition to the control wells, each well was added tBHQ with a concentration of 100μM. Each type of cell was repeated for five times. After a 24-h exposure to the compounds, the medium was removed and 100μg / ml EB 200μl was added to stain the cells for 20 minutes at room temperature. The cells were then washed with PBS once and 200μl PBS was added into each well prior to measuring flurescence. Measurement of GFP or EB fluorescence was performed using a fluorescence microplate reader respectively. The induced GFP expression level was the highest in HepG2-4ARE-TK-GFP cells and it was about 6 times compared to HepG2-TK-GFP. The clone cells with the highest GFP expression level and lowest level were used to do the follow-up experiments.4. After HepG2-TK-GFP and HepG2-4ARE-TK-GFP cell proliferation and inoculation into the black 96 well plates for 24 hours, different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. Each concentration was repeated for five times. After 24 hours of incubation, the cells were washed with PBS and stained with EB, fluorescence intensity of GFP and EB was measured by the fluorescence microplate reader. The results indicated that the GFP fluorescence of HepG2-TK-GFP cells was not induced by PDTC and tBHQ. The fluorescence intensity were irregular and the differences between concentrations were not significant (P>0.05). As the GFP fluorescence intensity increased with PDTC concentration in PDTC-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression was first increased and then decreased showing a dose-response relationship. At the concentration of 50μM, inducing effect had a high peak value and it was significantly different from the control (P<0.05). As the GFP fluorescence intensity increased with the tBHQ concentration in tBHQ-induced HepG2-4ARE-TK -GFP cells, the fluorescence expression continued to increase. There were significant differences between the experimental groups (12.5, 25, 50, 100, 200μM) and the control group (p<0.05) showing a dose-response relationship. Therefore, this transgenic cell model can be used to screen chemical compounds with different structural properties.5. A red fluorescent protein and promoter fragment was cloned into the vector pMD18-T from pDSRed2-N1 to construct the vector pMD-DsRed. The vectors pMD-DsRed and p4ARE-TK-GFP/neo were digested by Ade I and BspT1. The red fluorescent protein and promoter fragments were cloned into the vector p4ARE-TK-GFP/neo to construct the eukaryotic reporter vector p4ARE-TK- GFP/DsRed / neo. This vector was transfected into HepG2 cells. Positive cell clones were screened by G418 to construct the cell model HepG2-4ARE-TK-GFP/DsRed using GFP as the first signal and red fluorescent protein as the second signal. The cells were inoculation into the black 96 well plates for 24 hours and different concentrations (0 (DMSO control), 12.5,25,50,100,200μM) of positive tested compounds PDTC and tBHQ were added in. The results were consistent with the conclusions made for the HepG2-4ARE-TK-GFP cell model. Therefore, the transgenic cell model of HepG2-4ARE-TK-GFP/DsRed was successfully established and screening will become easier, more convenient and more accurate.6. The HepG2-4ARE-TK-GFP cell model was used to screen for 22 kinds of traditional Chinese medicine monomers, three types of active ingredients in Chinese medicine, Liu Wei Di Huang Capsule, Si Jun Zi Decoction, and ten single herb extracts. The results showed that andrographolide in Chinese medicine monomers, baicalin, rhein, emodin, quercetin, daidzein, ursolic acid, resveratrol were tested positive showing a dose-response relationship. Eight monomers could induce phase II enzymes expressions therefore played a chemopreventive role. Three effective parts of the total flavonoids in Scutellaria barbata and astragalus infection showed positive results, suggesting that their roles in blocking precancerous lesions and resistance to mutations may be achieved by the induction of phase II enzymes. The results for Liu Wei Di Huang capsule and Si Jun Zi Decoction were positive. Among single Chinese medicine extractions, the results were positive for Rehmannia glutinosa, ginseng, and Atractylodes. The effect of Liu Wei Di Huang capsule was better than the single herb Rehmannia glutinosa in the capsule, suggesting that the induced GFP expression in the model may be due to the new component produced or a combination of the compounds. This indicates that some natural chemopreventive agents was not less effective compared to some known synthetic chemical agents and more importantly, the side effect of these natural agents may be much smaller.