Study Of The Molecular Mechanism On Traditional Chinese Medicine Jinfukang For Intervention Of Lung Cancer Cells Based On High-throughput Approaches

Lung cancer is one of the most malignant cancers with high morbidity and mortality among all cancers in the world.Pathogenic causes of lung cancer mainly include smoking,heredity,profession,radiation,and so forth.At present,many therapeutic methods including surgery,radiotherapy,chemotherapy,immunotherapy,et al.,are used for lung cancer treatment.These therapeutic methods gave a great contribution for lung cancer patients for prolonging survival,relieving the pain and enhancing the quality of life in the past decades.But,these therapeutic methods still have defects during clinical usage.Traditional Chinese Medicine(TCM)has been used for lung cancer treatment for thousands years.Many studies demonstrated that multiple TCM formulas have anti-tumor efficacy.Jinfukang(JFK),an Astragalus-based herbal formula consisting of 12 herbs,has been reported to have the anti-tumor efficacy against Non Small Cell Lung Cancer(NSCLC).Although previous studies disscused the preliminary mechanism of its anti-lung cancer efficacy,the underlying mechanisms still need further discussion.Firstly,chromatographic and Mass spectrographic fingerprinting of JFK were detected by LC-MS.Morphological characteristics of nucleus was observed by DAPI staining.PI staining and flow cytometry were used for detecting the apoptotic cells.DNA damage was characterized by TUNEL assays.Secondly,holistic approaches(such as ChIP-seq and RNA-seq)were used for acquiring globally informations about protein-protein,protein-DNA interactions and gene expression from different samples.Responsed genes were screened out using bioinformatic analysis.Lastly,the methylation states and gene expression levels of responsed genes were verified via ChIP-qPCR and RT-qPCR respectively.Western blot was used for examining protein expression level.The biology function of responsed gene was verified by RNAi assay.The present study suggests that many signal pathways are involve in JFK-induced anti-lung cancer activity.On the one hand,we performed ChIP-seq assays to evaluate the effect of JFK through genome-wide profiling of H3K4Me3,an active modification marker for gene expression.Totally,our ChIP-seq analysis identified 11,670 genes with alteration of H3K4Me3 pattern(P < 0.05).Gene Ontology(GO)analysis indicates that these genes are involved in tumor-related pathways,including pathway in cancer,basal cell carcinoma,apoptosis,induction of programed cell death,regulation of transcription(DNA-templated),intracellular signal transduction,and regulation of peptidase activity.Particularly,we found that H3K4Me3 pattern at the promoters of SUSD2,CCND2,BCL2A1 and TMEM158 is significantly altered not only in A549 cells,but also in other lung cancer cell lines including NCI-H1975,NCI-H1650 and NCI-H2228 cells,after exposing to JFK.On the other hand,we found that JFK not only can induce lung cancer cells to apoptosis,but also could enhance DDP-induced apoptosis.Transcriptome profiling analysis indicated that combination of JFK and DDP regulates genes involved in apoptosis signaling pathway and MAPK signaling pathway.Bioinformatic analysis suggested that apoptosis-related genes including AIFM2,APOL1,PTPN6,CASP1,JUN,JUNB,MYC and TCF7L2 play an important role in the course of pro-apoptosis process,which activated by combination treatment with JFK and DDP on A549 cells.Moreover,we found the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines NCI-H1975,NCI-H1650 and NCI-H2228.AIFM2 knockdown assays demonstrated that AIFM2 mRNA and protein levels are up-regulated when A549 cells exposed to JFK and DDP simultaneously.Therefore,synergistic activation of AIFM2 contributed to the pro-apoptosis effect induced by combined treatment of JFK and DDP.Collectively,the present study performed the H3K4Me3 profiling in JFK-treated human lung cancer cell A549,and provided the first evidence for understanding the anticancer activity by modulating H3K4Me3 modification.These findings could hold promise for epigenetic diagnostic markers and drug targets for the treatment of human lung cancers.Importantly,this study performed the transcriptome prolifing in the A549 cells which treated with combination treatment of JFK and DDP,and gave the first evidence that activation of AIFM2 contributes to induction of pro-apoptosis by combined treatment with JFK and DDP in human lung cancer cells.Togehther,the results of the present study provide an insight for their potential clinical application in lung cancer treatment.