A Study Of High-throughput With High-content Screening For Anti-breast Cancer Natural Compounds

Breast cancer is one of the most common malignant tumors and the most common malignancy of women in the world. In our country, the incidence of breast cancer is increasing year by year. In big cities, breast cancer became the first carcinoma of women. Since current treatment modalities are inadequate, novel therapies are necessary to reduce the effects of the increasing incidence in lung adenocarcinoma. Despite the traditional treatment of breast cancer, such as surgery, chemotherapy and radiotherapy continue to be improved, breast cancer is still the leading death cause of women. Therefore, it is necessary to look for ideal drug to breast cancer. Traditional Chinese medicine (TCM) is a kind of natural products, and rich in resource, low in cost. In clinical practice, TCM show very little toxicity or side effects and have gained increasing attention as an important resource of screening new drug worldwide.High throughput screening (HTS) and High content screening (HCS) are the major techniques of drug discovery. HTS can perform automated testing, rapid acquisition and analysis of experimental data which is suitable for screening natural compounds; HCS can efficiently access the cell and the cell characteristics of the integrated cellular information, and become the most suitable screening method for integrated bio-pharmacological effects of drugs. The main problem restraining the HTS and HCS to use for screening the TCM is that there is no sample library suitable for these two methods. Particularly, HCS is not currently used in effective screening of TCM because of complex technique and high professional data processing requirements. Therefore, the establishment of TCM sample library to HTS and the screening of HCS to discover the antitumor compounds are currently new ideas of the drug screening.In present study, we chose 500 TCM to build up a compound fraction library. For construction of this library, HPLC was used for the separation of the TCM fraction. We select breast cancer MCF-7 cell line as the cell model to screen the potential anti-tumor compounds. We performed our HTS with modified MTT assay and the absorbance at 540nm (A540) was read on a spectrophotometric plate reader. The cell growth inhibition rate was calculated, and the IC50 values were obtained according the inhibition rate. The fraction whose inhibition rate was above 50% was defined as the positive. Preparative and analytical HPLC were used for the separation and purification of active compounds. The chemical structure of anti-cancer active compound was determined by MS and NMR. Further, the morphological change after drug treatment, the effect of active compound on the cell cycle and apoptosis was tested by HCS.The results show that a TCM compound fraction library including 40,000 fractions was successfully built up. In the library, each fraction contained less than 20 main component. On the basis of this library, we screened 1600 fractions and 1 TCM which can significantly inhibit the proliferation of MCF-7 cells. This drug is:Graden Balsam SeedSo far, Garden Balsam Seed were selected for the separation of the active compound because of its obvious inhibitory effect on the MCF-7 cells. Using extraction and analytical HPLC, we found that the retention time of the active fraction E3 in Garden Balsam Seed is 13.70min. After testing the toxicity of the compound on the mouse spleen cells, we found the compound has less toxic effect to normal cells. So we indentify the active compound by MS and NMR technology and the result is quercetin. By the HCS, we saw that the number of cells treated by Garden Balsam Seed is less than the negative control group. It indicated that quercetin can significantly inhibited the proliferation of MCF-7 cells. Further, it was found by HCS that the cells treated by Garden Balsam Seed were blocked at G2/M phase.In a word, the present study demonstrates that 1. The TCM compound fraction library for HTS was successfully built up.2. The successful separation of quercetin from Garden Balsam Seed proved our HTS system is feasible and credible;3. By HCS, quercetin was found to inhibit MCF-7 cells course from G1 to S, and then slow down cells proliferation.4. Quercetin can induce the apoptosis of MCF-7 cells; 5. Because Quercetin has the traits of low toxicity and strong inhibition, it will provide a new therapy for the breast cancer.