Screening Natural Small Molecule Inhibitor Of Non-Small Cell Lung Cancer And Studying Of Its Mechanisms

Cancer is a serious threat to human health, only in 2008, there were 12.7 million new cases worldwide and about 7.6 million people died from this desease. The incidence of cancer is rising daily with the rapid industrialization and urbanization and the number of people dying from cancer increasing year by year, most of all, there is increasing number of young patients suffering from cancers, which brings to a tremendous financial and emotional burden not only to the society but also to families.As one of 20 common cancers, lung cancer is becoming one of the most malignant cancers in the world. Lung cancer is on top of the incidence and mortality rate both in developed and developing countries. At present, there are 210 thousand new cases of lung cancer in China each year and the mortality rate is the number one among all cancers in cities of China. The incidence of lung cancer is rising daily and more and more yongers are involved in. According to the clinical pathology characteristics, lung cancer can be divided into two types:small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). NSCLC is the major one of lung cancer which accounts for 80%-85% lung cancer cases. In spit of the low malignancy and slow development, NSCLC brought a great deal difficulties to the clinical diagnosis and treatment for the threat of micro-metastasis in patients. According to statistics, more than 80% patients with advanced non-small cell lung cancer have lost the chances of receiving surgery treatment. Besides, compared with SCLC, NSCLC is much less sensitive to chemotherapy, consequently, the traditional chemotherapy can benefit little to patients of NSCLC.Chemotherapy is the standard treatment of advanced non-small cell lung cancer, but the current chemotherapy drugs with greater side effects and easiness to be tolerated benefits little to the NSCLC treatment, which retarded the outcomes of patients greatly. Therefore, new therapeutic drugs with more efficiency but lower side effects are urgently required. Traditional Chinese medicine(TCM) is a natural medicine, whose unique efficiency especially in the treatment of serious deseases was proved by the historical clinical practice in ancient ages. TCM has advantages over the traditional chemotherapy: such as TCM is rich in sources; has superior three-dimensional spatial structure; produces low toxicity; easy to adopt multi-drug combination to improve the therapeutic effect; low-cost in production and application, et al.However, TCM has unavoidable disadvantages defecting the useness of TCM in the treatment of neoplasms:the active components of TCM are difficult to be isolated; it is difficult to determine the efficacy of specific components; the action targets are not easy to definited; the side effects are unforeseen; there are traces of active components in TCM; the quality control of isolating of active components is difficult; TCM works slowly, et al. Therefore, separation and enrichness of effective components of Chinese medicine, definition of TCM targets, clarification of its mechanism are the intensive interests in the field of cancer treatment research with TCM. How to build techniques and methods system to efficiently separate active components from the complex natural medicines is the key.In this study, we take efforts to establish and improve an efficient system of methods and technical means for rapid separation of active components from TCM and use this system to screen and isolate new natural small molecules inhibitors of human non-small cell lung cancer. As a result, We sucessfully established an efficient method and technical means system of separation of effective components from TCM. Using this system, we smoothly obtained 42 Chinese herbal medicines with significant inhibitory activity on A549 from the fraction library of 500 Chinese medicine by whole-cell based high-throughput screening method.252 fractions distributed in 44 fractions group (cluster) are involved. The further assays were carried out to assess the toxicity of these positive fractions aganist mouse primary spleen cells. Consequently, up to 20 species of Chinese medicine including 228 components were found low toxic to the primary mouse spleen cells.Another task of this study was to screen natural single compounds with inhibitory effects on NSCLC.400 natural monomer compounds have been chosen to examine the activity on A549 cell by whole-cell based high-throughput screening and 19 natural single compounds were found to be strong inhibitors of A549 cells, alantolactone was one of them.This study furtherly divided active fraction of Bupleurum to single compounds, ultimately, the inhibitory single compound NSL (A549)-BUP was obtained. The analysis of molecular structure of NSL (A549)-BUP by NMR and mass spectrometry revealed that this compound was one of Terpenoids with the molecular weight of 781 Dalton. The analysis results of inhibition mechanism showed that NSL (A549)-BUP efficiently induced A549 cell apoptosis, arrested A549 at G2/M phase. Additionally, the apoptosis-inducing effect of alantolactone was found in A549 cell line. Cell cycle arresting assay also clarified that this small molecule compound retarded A549 cycle at the G0/G1 and S phase.This study successfully established the system for isolation of active single compounds from complex natural medicine for treatment of NSCLC. A number of small molecular natural compounds inhibiting A549 were obtained from fractions library of 500 Chinese medicine and the monomer compound library, which paved the way of obtaining high efficiency yet low toxicity of drugs for the treatment of advanced NSCLC. Meanwhile, the results of this study lay the foundation for choosing specific natural molecular for targeted therapy of NSCLC. The obtained small molecule inhibitors may be used as powerful chemical biology tools to study the mechanisms of the occurrence, development and metastasis of lung cancer.