Herbal Formula YYJD Induces Lung Cancer Cell Apoptosis Via Activation Of EGR1

Objective Yangyinjiedu fomula(YYJD)as an optimization of clinical effective Chinese herbal compound “Jinfukang” in China,there are 5 herbs in YYJD with effect of supplementing Qi and nourishing Yin,clearing heat and removing toxicity.Early research showed that YYJD is an effective and less toxically Chinese compound in vitro and in vivo.However,the underlying mechanisms remain to be investigated.In this study,we examined the anticancer activities of Chinese herbal formula Yangyinjiedu(YYJD)and found YYJD inhibits lung cancer cells viability.Presently,little is known about the potential mechanism of YYJD and our studies aim to elucidate its mechanism in lung cancer treatment.Methods 1.The effect of YYJD on the biological behavior against lung cancer cells in vitro.CCK-8 was used to detect the growth inhibition effects of YYJD on human non-small cell lung cancer cell lines(A549,NCI-H2228,NCI-H1299,NCI-H1975,NCI-HCC827)and mouse lung cancer cell line(Lewis,LLC)at different concentrations and different time.The sensitive cell lines of YYJD were obtained.The effects of different concentrations of YYJD on the cell cycle of human non-small cell lung cancer cell lines(A549,NCI-H2228,NCI-H1299)were detected by flow cytometry.The effects of different concentrations of YYJD on apoptosis of human non-small cell lung cancer cell lines(A549,NCI-H2228,NCI-H1299)were detected by Hochest staining,flow cytometry,Respectively.2.Characterization of the YYJD-induced differential gene expression pattern in lung cancer cells.To understand the underlying molecular mechanisms of the growth inhibition and apoptosis effects induced by YYJD,we next performed a transcriptome analysis to investigate differential gene expression in YYJD-treated A549 through RNA-seq;Using DAVID we next performed gene ontology(GO)analysis with differentially expressed genes upon YYJD treatment 3.To further verify the functional relevance of EGR1 activation in YYJD-induced apoptosis,we performed si RNA assay to suppress EGR1 expression.si EGR1 knockdown cells were treated with YYJD(63 μg/ml)for 48 h,CCK-8 and flow cytometry were used to detect the inhibition of lung cancer cell proliferation and induce apoptosis by silencing EGR1.4.To understand how EGR1 exerts its pro-apoptosis activity through modulating its downstream target genes.To this end,we performed chromatin immunoprecipitation(Ch IP)coupled with deep sequencing(ChIP-seq)analysis to interrogate EGR1 binding targets across the whole genome in YYJD-treated A549 cells.To understand how EGR1 affects the expression status of its downstream target genes in YYJD-treated lung cancer cells,we analyzed the EGR1-associated transcription network by intersecting ChIP-seq data with the transcriptome data.5.we generated lung cancer cell tumor xenografts,which were subsequently treated with YYJD,cisplatin,respectively or in combination.We found both YYJD and cisplatin inhibit the growth of tumor xenografts.6.In vivo,two different strains of mice were intragastrically administered by YYJD.After 30 days of intervention,blood samples were taken for liver and kidney function,and important organs were weighed to calculate organ index.HE staining was used to detect the pathological damage of organs.Results 1.The studies showed that YYJD could decreased the viability of various lung cancer cells(A549,NCI-H2228,NCI-H1299,NCI-H1975,NCI-HCC827,LLC)in a time-and concentration-dependent manner;we found treatment of A549,NCI-H2228,and NCIH1299 cells with YYJD resulted in a significant increase in the proportion of cells at the G2/M phase(* P <0.05),The higher the concentration,the stronger the blocking effect(△P <0.05);We observed that YYJD treatment triggered lung cancer cells exhibiting apoptotic morphology with condensed nuclei membrane blebbing,vacuolation in the cytoplasm and formation of apoptotic bodies the by Hochest 33342 staining;It was further confirmed by flow cytometry that the total apoptotic cells were significantly increased(* P < 0.05)in dose-dependent manner upon YYJD treatment for 48 hours in A549,NCI-H2228 and NCI-H1299;2.Transcriptome analysis showed that compared with the control,2,178 differentially expressed genes(P<0.05)were identified,with 1,464(67.2%)up-regulated and 714(32.8%)down-regulated,the top twenty GO terms,including cell proliferation-and cell death-related processes.In particular,we did observe that apoptosis-related process is significantly enriched among these GO terms.Among the differentially expressed genes EGR1 was most up-regulated;Western blot results showed that EGR1 activation was also observed in lung cancer cell lines A549 NCI-H2228 and NCI-H1299 with YYJD treatment.3.Although EGR1 is activated upon YYJD treatment,it is remarkably suppressed by si RNA both in m RNA and protein levels.Moreover,we observed a considerable decrease in both viability inhibition and pro-apoptosis activity in YYJD-treated A549 cells when EGR1 is suppressed.These results suggest that EGR1 is involved,in part at least,in YYJD-induced apoptosis in lung cancer cells.4.There were 1,697 binding sites(-5 ～ +2 kb)in A549 cells with YYJD.To identify EGR1-bound target genes.To understand how EGR1 affects the expression status of its downstream target genes in YYJD-treated lung cancer cells,we analyzed the EGR1-associated transcription network by intersecting ChIP-seq data with the transcriptome data.As mentioned above,we found YYJD induced 2,178 genes to be expressed differentially(P < 0.05).Among these genes 275 genes are bound by EGR1,with 193 upregulated and 82 down-regulated.Thus,these EGR1-bound and YYJD-responsive genes are potentially involved in biological activities we observed in YYJD-treated lung cancer cells.To verify this assumption,we performed gene ontology(GO)analysis of EGR1-bound and up-regulated genes with DAVID.Consistent to our observation,we did find that some up-regulated genes were enriched in GO terms positive regulation of apoptotic process,including ABR,ING2,CYP1B1,HIP1 R,KLF11,VAV2,TGFB1,BCL2L11 and DUSP6.5.The Lewis cell C57 BL/6 mouse subcutaneous xenograft model was used.Compared with the control group,YYJD(18.8 g/kg)could significantly inhibit the growth of the tumor.When combined with cisplatin,the anti-tumor effect of YYJD was stronger than that of YYJD or Cisplatin alone.(*P<0.05,**P<0.01,***P < 0.001);HE staining results showed that YYJD can effectively induce tumor cell death;we observed the obvious increase in protein level of EGR1.In addition,we found an apoptosis-related gene KLF11,BCL2L11,which were EGR1 downstream targets identified in this study,is also activated upon YYJD treatment.6.In vivo studies showed that YYJD had no significant effect on mice body weight,liver and kidney function,and organ index of normal mice.Conclusion 1.This study confirmed that YYJD can effectively prevent the growth of lung cancer and induce apoptosis of lung cancer cells in vitro and in vivo.2.This study introduced high-throughput omics technology and found that the anti-lung cancer mechanism of YYJD is related to the activation of transcription factor EGR1 and its downstream genes to induce apoptosis.This study provided a rich micro-material basis for the prevention and treatment of lung cancer.The theory and scientific connotation of treating phlegm with Yiqi Yangyin method.3.The method of transcriptome sequencing combined with ChIP-seq can be used to analyze the mechanism of multi-target anti-tumor of traditional Chinese medicine compound,and provide a methodological basis for further explanation of the mechanism of action of compound Chinese medicine.4.This study confirmed that YYJD with the effect of supplementing qi ang nourishing yin had small toxicity and safety.