The Screening And Preliminary Mechanism Research Of Synthetic Compounds For The Growth Inhibition On Lung Cancer Cells

Recently for better understanding the developmental mechanisms of human diseases including cancers, the researchers have been in a bottleneck situation to find the novel effective drugs. It was very impossible to explore a cancer developmental process only using either the biological or chemical approaches. In recent years, huge advance in Chemical Biology, a joint discipline of biology and chemistry has greatly helped the drug development. Chemical screening using synthetic small molecule libraries has provided a huge amount of novel active molecules which could act as the lead drug with their structural diversity. It establishes a foundation for mechanistic investigations on intangible biological processes.Lung cancer has been one of the leading causes to endanger human health seriously. There are lots of side effects with the present drugs for their specificity on cancer cells. In recent decades, specific cancer cell targeted drugs have been developed and a major trend of drug development. In this study, we identified two chemical compounds,22-H7 and 30-G5, through chemical biological screening using A549 cells against a structurally novel and diverse synthetic small molecule library of 1680 compounds. Both compounds harbored strong anti-proliferation activity on the non-small cell lung cancer cell A549, on the basis of with the IC50 values of 4.95±0.40μM and 0.55±0.05μM, respectively.The compound 22-H7,8-[(E)-2-[(4-bromophenyl)methylidene] hydrazin-1-yl]-3-methyl-7-(2-phenoxyethyl)-2,3,6,7-tetrahydro-1H-purine-2,6-dione, is a kind of purine-dione. and a structure-activity relationship was conducted with its 17 structural analogs. We found that 22-H7 was the only active compound which contained a bromo group. The result revealed that-Br acted an irreplaceable role in the process of inhibiting the growth of A549 cells. We then showed that 22-H7 treatment could induce the nuclear shrinkage and result in apoptosis in the A549 cells using the Hoechst and Annexin V/PI staining. The PI staining displayed that there is not any arrest during the cell cycle process. Futher, we have demonstrated that 22-H7 induces the depolarization of mitochondrial membrane potential and an increase of caspase-8 and-3/7 activity in A549 cells.Our results demonstrated that the apoptosis induced by 22-H7 was through death-receptor pathway possibly.With the survival detection on the four cell lines, we found that after treatment with 30-G5, N-butyl-2-methyl-4.9-dioxo-4.9-dihydronaphtho [2,3-b] furan-3-carboxamide, the growth inhibition of the non-small cell lung cancer cells A549 (IC50=0.55±0.05μM) was stronger than the human breast cancer cells MCF-7 (ICso=1.21±0.21μM). And there is little growth inhibition on the human normal embryo lung fibroblast cells MRC-5, but the IC50 value on the human nomal mammary epithelial cell was 2.06±0.17μM. The result showed that 30-G5 has some specificity and targeting properties during the process of growth inhibition, and its significance on the clinical therapy for lung cancer was much better than breast cancer. Further, we showed that 30-G5 treatment increased the number of Annexin V-positive staining cells, but not G1 or G2/M phase arrest in the cell cycle after PI staining. The JC-1 probe loading revealed that 30-G5 treatment induced a decrease of the mitochondrial membrane potential, but not an increase of caspase-3/7,-8 and-9 activities in A549 cells. Apoptosis inducing factor (AIF) play a critical role during the caspase-independent pathways, and then we used the small interfering RNA against the expression of AIF. We identified the survival rate of A549 cells by the MTT assay after silencing effect of AIF-SiRNA. The result showed that AIF-SiRNA could reduce the death of A549 cells induced by treatment with 30-G5. The western blotting revealed that AIF released from mitochondria into cytoplasmic and transferred to nuclear after treatment with 30-G5. The assays of ROS and Ca2+　level in A549 cells revealed that 3O-G5 treatment did not induce the change of the concentrations of ROS and Ca2+.Our results demonstrated that AIF has an important roles during our chemical growth inhibition against A549 cells, and the release from mitochondria into cytoplasm and the nuclear translocation were not linked with ROS and Ca2+.In conclusion, this study has identified two novel compounds which owned anti-proliferative activities on lung cancer cell A549. They could induce apoptosis with different pathways and provided potential possibilities against lung cancer.