Expression Profile-based In Silico Screening Of Candidate Therapeutic Compounds For Lung Adenocarcinoma And Related Experiments

Lung cancer, including small cell lung cancer and non-small cell lung cancer((NSCLC), is the major killer for the health of humans in the world,also the leading cause of cancer death for both men and women in China.Lung adenocarcinoma,belonging to NSCLC,is the predominant histological subtype of lung cancer and also is the most common kind of lung cancer, accounting for about 20-30% of primary lung cancers.At present, the therapeutic methods for NSCLC in clinical are usually surgical therapy, radiation therapy and chemotherapy,etc. Although the rapid development of the therapy for NSCLC was obtained, the outcome of patients has not improved significantly.The five-year survival rate is about 10% for most NSCLC patients including lung adenocarcinoma。Thus, there is need for some additional strategy aimed at the disvover of another effective therapy drugs for lung adenocarcinoma in order to prolong the life and reduce the mortality of lung adenocarcinoma patients.The gene-chip (bio-chip) is major technique in bioscience field in the 21st century, has the outstanding of high-throughput and rapid detecting,etc. Gene expression profiling chip has the superiority on the study of gene expression profiling mode of cells,which can be used to study the change of gene expression profiling in every stage of tumor cell develop,understand the gene expression mode during the occurrence and development of tumor.Thus,gene expression profiling chip has widespread application in tumorigenesis, early diagnosis, tumor genotyping,directing therapy and prognosis evaluation,etc. With the rapid development and widespread application of gene expression profiling chip, redundnt and complicate biology data was bring out. How to analyze tens of thousands gene hybridization information on gene-chip to bring to light vital signs and rule,which has become the major "bottleneck" restricting the further development of gene expression profiling chip. The analysis of tumor expression profiling chip is useful for the reveal of the mechanism of tumorigenesis,the diagnosis and therapy of tumor. Bioinformatics is newly rising subject which elucidates and reveals the biological meaning hided in much data set. Bioinformatics provids a new approach for the analysis of DNA microarray data,also become a new tool for the discovery of the drug and speed up the process of drug discovery.The discovery of drug experience several stages including the discovery of drugs from the nature,the discovery of drugs based on the mechanism of diseases,and drug discovery based on targets.The process of drug discovery also is from the blind drug discovery to the rationality drug design.At present,the discovery of drugs enters a new era, i.e,the discovery of drugs based on disease target-genes. Recently, many researcher created gene expression profiling databanks based on active compounds or drugs,amonge Lamb et al and his coworkers created a searchable database ("connectivity map") of the gene-expression profiles of human cells, cultured in vitro and exposed to diverse small molecules.C-MAP represents a tool for the large-scale discovery of unexplored connections among small molecules, diseases, and the biologic pathways that join them. Based on global gene expression profiles of disease, C-MAP not only provided a new way to the discovery of drugs,but also created a new mode of drugs'discovery,i.e, gene expression profiles-based drug discovery, which can be used for the screen of therapeutical candidate compounds for diseases, especially orphan diseases.Now, Lamb's method have been used by many researcher and proved helpful in potential therapeutic drugs discovery.To sum up, since lung adenocarcinom, one of the most common kind of lung cancer,become the major threaten for the health of human, there is imminent need for some additional strategy aimed at the disvover of another effective,litte adverse reaction drugs for lung adenocarcinoma.In the study,based on the mode of gene expression profiles-based drug discovery approach,we set out to discover agents for lung adenocarcinoma with computational and system biology tool "connectivity map",which provided the theory and experiment proof for the combined therapy of lung adenocarcinoma,and also accelerate the discovery of drugs for lung adenocarcinoma.The study includes the following three parts:PartⅠgene expression profiling based approach identifies candidate therapeutic compounds for lung adenocarcinomaObjective:To explore gene expression profiles-based approach for the screening candidate therapeutic compounds against lung adenocarcinoma,and screen the candidate compounds for lung adenocarcinoma.Methods and Results:1.Acquisition lung adenocarcinoma.public microarray data from GEO databank Raw data(.Cel files) of two published microarray data (gse7670 and gse10072) used in the study was obtained from Gene Expression Omnibus(GEO) website with the Ftp download tool.Amonge gene datasets GSE 7670, containing 64 samples, from Taipei veterans hospital;GSE10072 containing 107samles from the genetic epidemiology department of N.I.H.two datasets were based on GPL96 platform.The chip quality analysis was performed on all samples with the soft RMAexpress to reject disqualification microarray samles,we obtained 36 qualified samles(16 adenocarcinoma vs 20 normal)from GSE7670,43 samles(20 adenocarcinoma vs 23 normal)from GSE7670.2.Meta-gene analysis of lung adenocarcinoma Meta-gene analysis of lung adenocarcinoma vs normal were alone performed with the software packages BRB Array Tools, A total of 2,000 permutations were completed to identify the list of probe sets with a false discovery rate of<10% at a confidence of 95%.Differential expression was considered significant at 2-fold change.Finally, we obtained 344 differentially expression gene, amonge 93 up-regulated gene and 251 down-regulated gene3. The screen of drugs for lung adenocarcinom with Connectivity Map analysisFor the c-Map analysis, we created a gene query signature based on above common differential probe IDs.On c-Map analysis online,multiple drugs were identified to revert the signature of lung adenocarcinoma,including the following 4 categories:1)HSP90 inhibitors(tanespimycin, monorden,and alvespimycin);2) HDAC inhibitors(trichostatin A, vorinostat);PPAR receptor agonist(15-delta prostaglandin J2,troglitazone, pioglitazone,bufexamac)and PI3K inhibitors (LY-294002)Conclusions:Our study showed that gene expression profiles-based approach is feasible for screening lung adenocarcinoma candidate therapeutic compounds.Through cMAP analysis,we discoved some candidate compounds for adenocarcinoma, which can be further validated throuth experiment。 Part II Inhibition of 17-AAG on proliferation of lung adenocarcinoma cell line A549Objective:Multiple candidate compounds for adenocarcinoma,especially including some Hsp90 inhibitors,were screened out in part I, amonge 17-AAG, a chemical derivate of geldanamycin,obtained high negative enriching score.Here,we explore the inhibition of 17-AAG on the proliferation of lung adenocarcinoma A549 cell,and study the effect of 17-AAG on cell cycle and cell apoptosis of A549 cell.Methods and Results:1.Inhibition of 17-AAG on proliferation of lung adenocarcinoma A549: Inhibition of 17-AAG on proliferation of A549 was evaluated with MTT assay.The study were divived into different dosage of 17-AAG(0.2,0.4,0.8,1.6 and 3.2μM),negative control and bland control.After A549 cell were treated with above various dosage 17-AAG for 24,36,and 48h, MTT assay were performed.Our result showed that 17-AAG could inhibit the proliferation of A549 cell line on dose-dependece mode,there have statistics difference in different dose group (F=586.02,P=0.000).There exist reciprocation effect between dose and time (F=18.789,P=0.000).The IC50 value (Median Inhibition Concentration) at 24,36,48h,were 1.539,0.884 and 0.455μM,respectively, which indicated that 17-AAG could inhibit the proliferation of A549 on time-dependece mode.2.The effect of 17-AAG on cell cycle of A549 cell. After A549 cell were treated with various dosage 17-AAG for 24, cell cycle of A549 were analyzed by flow cytometry. Our present study demonstrated that 17-AAG caused the G2/M block of A549.The G2/M cell ratio were(12.93±0.29)% in 0.11μM group, (20.57±0.29)% in 0.23μM group, (23.00±3.64)% in 0.45μM group,respectively, showing statistic difference when compared with that of in control group (6.03±0.95)% difference(P<0.001).3. The effect of 17-AAG on the apoptosis of A549 cell After A549 cell were treated with various dosage 17-AAG for 24, the apoptosis of A549 were detected with Annexin V/PI staining. Our study indicated that 17-AAG induced the apoptosis of A549 on dose-dependece mode. The early apoptosis ratio in 0.11μM 17-AAG have no statistic difference when compared with control group(P>0.05).The advanced apoptosis rates of A549 in 0.23μM 17-AAG group was 11.86±0.951% have statistic difference.(P<0.05).The early and advanced apoptosis ratio in 0.45μM 17-AAG was 9.35±0.574% and 21.316±1.036%,respectively, higher than that of control group, 0.11μM and 0.23μM 17-AAG group.(P<0.005).Conclusion:17-AAG can inhibit the proliferation of A549,cause the G2/M block of A549,and induce the apoptosis of A549.Part III Effect of 17-AAG combined with cisplatin on proliferation of lung adenocarcinoma A549 cell lineObjective:Cisplatin is the common chemotherapeutic drug for nonsmall-cell lung cancer(NSCLC),but have some shortage of more adervse reaction,toxicity and drug resistance,etc。Thus, there is need for the discovery of some effective,litter side effect drugs which can enhance the antitumor effect of cisplatin. Our study in part II demonstrated that 17-AAG can inhibit the proliferation of A549,cause the G2/M block of A549,and induce the apoptosis of A549.Here, we would discuss the effect of 17-AAG combined with cisplatin on proliferation of A549,which will provid more experiment proof for the further clinical applicaton of 17-AAG.Methods and Results:1.Inhibition of 17-AAG combined with cisplatin on proliferation of A549: The study were divived into different dosage of 17-AAG(0.025,0.05,0.1,0.2 and 0.4μM),various of cisplatin(4.4,8.7,17.5,35and70μM),and 17-AAG+cisplatin. A549 were treated with drugs for 48.We evaluated the effect of 17-AAG combined with cisplatin using the method of Chou and Talalay. The study showed that there existed synergistic action when 17-AAG combined with cisplatin. The Dm value of cisplatin or 17-AAG reduced when 17-AAG combinated with cisplatin, Dm value of cisplatin is 69.627μM for single,16.19μM for combination(reducing 4.3 times);that of 17-AAG is 0.4547μM for single,0.093μM for combination(reducing 4.89 times). We ploted the relationship between the effect(fa) and combination index(CI) when 17-AAG combinated with cisplatin。With the dose increase of 17-AAG and cisplatin, the value of fa and CI also increases.When fa>0.8,CI>1,meaning antagonistic effect;hower when fa<0.8,CI<1,showing synergistic effect,i.e, synergistic effect is obtained when 17-AAG(<1.24μmol/L)combinated with cisplatin (<217μmol/L).2. The effect of 17-AAG combinated with cisplatin on cell cycle of A549. After A549 cell were treated with various of 17-AAG(0.11,0.23,0.45μM)+70μM ciplatin,cell cycle were analyzed by flow cytometry. Our study indicated that there are no between the cell rates of G1 or G2/M stage in various 17-AAG combination with ciplatin group and that in the control group,only that of S stage in 0.45μM17-AAG+70μM cisplatin group have slight increase, showing statistic difference when compared with that in the control group(P<0.05).3. The effect of 17-AAG combinated with cisplatin on the apoptosis of A549 After A549 cell were treated with 17-AAG or ciplatin or 17-AAG+ciplatin combination for 24, the apoptosis of A549 were detected with Annexin V/PI staining. Our results showed that there have statistic difference in the early rates(F=97.60, P=0.000)and advanced apoptosis rates (F=645.01,P=0.000).The early and advanced apoptosis rates of A549 in 0.11μM 17-AAG+70μM cisplatin group have no significant increase when compared with that of 0.45μM 17-AAG or 70μM cisplatin group(P>0.05).However, The early and advanced apoptosis rates of A549 in 0.45μM 17-AAG+70μM cisplatin group have significant increase when compared with that of 0.45μM 17-AAG or 70μM cisplatin or 17-AAG (0.11 or 0.23μM)+70μM cisplatin group(P<0.005).Conclusion:Our finding show that there have synergistic effect when 17-AAG combined with cisplatin.