Identification Of New Natural Compounds With Anti-lung Adenocarcinoma Activity By Hith Throughput Screening From Chinese Medicinal Herbs

Lung cancer is one of the most cancers and the leading cause of cancer death in the world. Recently, its incidence and mortality showed a clear upward trend. In 2002, there were 1,350,000 new cases of lung cancer in the world, and 1,180,000 people were dead. In China, lung cancer has the highest mortality rate among all the cancers in the city dwellers. In 2005, there were 540,000 new cases of lung cancer in China, and 480,000 people were dead. The major patients of lung cancer are non-small cell lung cancer (NSCLC), which comprises approximately 80% of lung cancer patients. In recent years, the incidence of lung adenocarcinoma, a subtype of NSCLC, is increasing and becoming the most subtype of lung cancer in Asian patients. Such as surgery, radiotherapy and chemotherapy, the conventional treatment of lung cancer have provided some new avenues for therapeutic strategies. However, certain patients are still plagued by rapid disease recurrence and progression. Particularly, lung adenocarcinoma is often at the stages beyond surgical remedy and resistant to chemotherapy and radiation. There has been no significant improvement in the overall survival. The 5-year survival rate of lung cancer is less than 15% in U.S.A and 9% in developing countries. The chemotherapeutic benefits and the prognosis of lung adenocarcinoma remain to be promoted. Thus, the development of new anti-lung adenocarcinoma drugs becomes more important and urgent issue.Traditional Chinese medicine (TCM) is a kind of natural products, and generally low in cost, rich in resource. In clinical practice, TCM has been prescribed for many diseases over centuries and show very little toxicity or side effects. Recently, TCM began to be matched by increasing scientific attention as an important resource of screening new drug worldwide. However, despite the vast interest and increasing demand, the complexity of TCM and the lack of credible data for its mechanism are the main obstacles toward the treatment for many diseases, such as cancer. High throughput screening (HTS) is the major technique of drug discovery. Drug library is the important part of HTS. Compared with combinatorial chemistry compound and derivatives of proved drugs, the natural compound library shows many great advantages such as plentiful and structural diversity. However, there is no such an ideal herbal-based natural compound library that has good therapeutic coverage and is suitable for HTS. It is necessary to create such sample database of TCM and find anti-cancer drugs from it.Our strategy is to build up a TCM compound fraction library from a collection of most frequently used 500 TCM according hydrophilic. The library consisted of 40,000 fractions from Chinese traditional medicines. For construction of this library, HPLC was used for the separation of the TCM fractions. We select lung adenocarcinoma A549 cell line as the model to screen the potential anti-tumor small molecules. We performed our HTS with modified MTT assay, and the absorbance at 540nm (A540) was read on a spectrophotometric plate reader. The proportion of surviving cells and he inhibition rate were calculated. The IC50 values were obtained according the inhibition rate. The fractions 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. Mass spectrometer (MS) and nuclear magnetic resonance (NMR) techniques were selected to determine the chemical structure of anti-cancer active compound. The toxicity of active compound was detected by MTT method on A549 cells and human peripheral blood leukocytes. Hoechst33258 staining was used to find out the morphological changes after drug treatment. The effect of active compound on the cell cycle was analyzed by flow cytometry.The results indicated the TCM compound fraction library including 40,000 fractions was successfully built up, in which each fraction contained less than 20 main components. On the basis of this library, we screened 16,000 fractions from 200 TCM and found 24 fractions of 8 TCM with the ability of inhibiting to the growth of A549 cells. These herbs are:Thinleaf Milkwort Root, Sharpleaf Galangal Fruit, Malaytea Scurfpea Fruit. Sevenlobed Yam Rhizome, Lotus Rhizome Node, Blighted Wheat, Garden Balsam Seed and Selaginella tamariscina. The toxicity test of these drug fractions on the mouse spleen cells showed that Garden Balsam Seed and Selaginella tamariscina are less toxic to spleen cells. So far, Garden Balsam Seed and Selaginella tamariscina were chose for the separation of the active compound. The analytical HPLC showed that the retention time of the active fraction E3 in Garden Balsam Seed is 13.75min. MS and NMR technology identified that the active compound is quercetin. The IC50 value of quercetin against A549 cells is 10.8umol/l. By the same method, the analytical HPLC showed that the retention time of the active fraction G8 in Selaginella tamariscina is 25.80min. MS and NMR technology identified that the active compound is ALA. The IC50 value of ALA against A549 cells is 10.2umol/l. Many articles have described the anti-cancer mechanism of quercetin, but less about ALA. So far, we further studied the anti-cancer mechanism of ALA by morphological observation and flow cytometry. We found that the IC50 value of ALA against human peripheral blood leukocytes is 62.5umol/l. This result indicated ALA has little toxic effect on peripheral blood leukocytes. By Hoechst staining, we found apparent apoptosis bodies in treatment group. At the same time, flow cytometry showed that the cell cycle of A549 treated by ALA is blocked in G2/M phase. These results suggested that ALA can induce apoptosis for A549 cell. In addition, the separation and identification of other 6 active compounds were in progress.Taken together, the present study demonstrates that 1. The TCM compound fraction library for HTS was successfully built up.2.24 active anti-cancer ingredients of 8 TCM were found from our library; 3. The successful separation of quercetin from Garden Balsam Seed proved that our HTS system is feasible and credible:4. The anti-cancer active compound ALA was separated from Selaginella tamariscina for the first time; 5. ALA can inhibit A549 cells course at G2/M phase, then slow down cells proliferation.6. ALA can induce the apoptosis of A549 cells; 7. Because ALA has the traits of low toxicity and strong inhibition, it will provide a new therapy for the lung adenocarcinoma; 8. The separation of other 6 active compounds is in progress.