Investigation Of Small Molecule-induced Apoptosis And The Molecular Mechanisms In A549 Human Lung Cancer Cells

INVESTIGATION OF SMALL MOLECULE-INDUCED APOPTOSIS AND THE MOLECULAR MECHANISMS IN A549 HUMAN LUNG CANCER CELLSIn recent years,lung cancer is the leading category of cancer threatening the health of the people in many countries,lung cancer mortality stands the first within all kinds of cancer death.Moreover,among all types of lung cancer,adenocarcinoma occures most commonly and its incident rate is the highest.More and more studies evident that cell apoptosis inhibition is the major cause of tumorigenesis and progresss.Therefore,induction of apoptosis by drugs in lung adenocarcinoma cells is an indispensable strategy for lung adenocarcinoma therapy,which would decrease lung cancer mortality.In this article,we aimed to screen small molecules to effectively promote lung adenocarcinoma A549 cell apoptosis by using Chemical Genetics,the novel technology platform.Besides,we also aimed to investigate the mechanisms by which the small compounds function.This study would lay the foundation for us to insight into the proteins targeted by the small molecules,and would provide a theoretical basis for developing anti-lung adenocarcinoma agents.STUDY CONTENTS:1 Screening the small molecules initially.2 Identification of apoptosis induction in A549 cells by small molecules.3 Study the mechanisms of the small molecules inducing apoptosis in A549 cellsMETHODS:1.MTT assay for cell viability,by which small molecules were screened initially.2.The morphological changes were observed under phase contrast microscope.3.LDH assay to determine whether the cells treated by the small molecules undergo necrosis.4.DNA nuclear fragmentation was analyzed by acridine orange staining.5.The TdT-mediated dUTP nick end labeling technique was used to detect in situ nuclear DNA fragmentation and count apoptosis ratio6.The fluorescence probe,DCHF was used to analyze ROS level.7.The change of the mitochondrial membrane potential was measured by the TMRM staining as described previously[Falchi et al,2005]8.The expression of integrinβ4 was analysised by immunofluorescence assay.RESULTS:1.The effects of 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives on A549 lung cancer cell growth1.1 The results of MTT assay showed that 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives(1-5~#)could inhibit A549 lung cancer cell growth within 24-48 h,and in a time and dose dependent manner.1.2 Cell morphological changes observed under Phase Contrast Microscope:When cells were exposed to 2H-benzo[b][1,4]oxazin-3(4H)-one derivatives(1-5~#) 200μM for 24h,morphological changes were found,including cell shrinkage, round and cell viability decreased.2.The effects of 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives on A549 lung cancer cell growth2.1 The results of MTT assay showed that all kinds of six 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives(8a-8f)could decrease cell viability markedly within 24-48 h,and in a time and dose dependent manner.2.2 Cell morphological changes observed under Phase Contrast Microscope:When cells were exposed to 8d-8f 20μM for 24-48h,morphological changes which exhibiting the classical characteristics of apoptosis occurred.2.3 Nuclear fragmentation assay by AO staining in conjunction with laser scanning confocal microscopy(LSCM)showed that when incubation with 8d-8f 20μM for 48 h,many cells showed red-orange nuclear fragments and apoptotic bodies.2.4 LDH assay showed that when the cells were incubated with 8d-8f 20μM for 48 h,there was no significant difference(p＞0.05)in LDH release between the control group and the test group.2.5 TUNEL assay showed that the 8d-8f 20μM and 48h treatment group showed an overwhelming majority of apoptotic A549 cells(P＜0.01).2.6 When A549 cells were treated with 8d-8f 20μM for 48h,the intracellular ROS level was decreased obviously(P＜0.01).2.7 When A549 cells were treated with 8d-8f 20μM for 48h,the mitochondrial membrane potential level was decreased obviously(P＜0.01).2.8 The results ofβ4 protein expression detected by immuno-fluorescence assay combined with LSCM showed that when A549 cells were treated with 8d-8f 20μM for 48h,the relative level ofβ4 was up-regulated markedly(p＜0.01).CONCLUSION:1.Based on the initial screen of the small molecules,we concluded that all of the five kinds of small molecules could inhibit the proliferation of A549 human lung cancer cells,but their abilities in proliferation inhibition were quite different.2~#,3~# and 5~# were the most powerful.2.The results of MTT assay showed that all kinds of six 5-alkyl-2-ferrocenyl-6,7-dihydropyrazolo[1,5-a] pyrazin-4(5H)-one derivatives(8a-8f)could decrease cell viability markedly,but their abilities in proliferation inhibition were quite different.8d,8e and 8f were the most powerful,so our research was mainly focused on the effects of 8d-8f on the proliferation of A549 cells and the mechanisms by which the small compound functioned.3.8d,8e and 8f,20μM,could induce apoptosis in A549 human lung cancer cells. They might perform their functions by up-regulation of ROS level and effect the mitochondrial membrane potential level.4.Integrinβ4 level was up-regulation also,suggesting that integrinβ4 might be a key factor in the signal apoptosis.