Herbal formula Yangyinjiedu induces lung cancer cell apoptosis via activation of early growth response 1

Abstract

Traditional Chinese Medicine (TCM) has been extensively used in clinical practices and proven to be effective against cancer. However, the underlying mechanisms remain to be investigated. In this study, we examined the anticancer activities of Chinese herbal formula Yangyinjiedu (YYJD) and found that YYJD exhibits cytotoxicity against lung cancer cells. Transcriptome analysis indicated that 2178 genes were differentially expressed (P < 0.05) upon YYJD treatment, with 1464 being (67.2%) up-regulated. Among these, we found that the tumour suppressor early growth response 1 (EGR1) is the most activated. We demonstrated that EGR1 contributes to YYJD-induced apoptosis in A549. Through dissecting EGR1-associated transcriptional network, we identified 275 genes as EGR1 direct targets, some targets are involved in apoptosis. Lastly, we observed that YYJD enhances EGR1 expression and induces cell death in tumour xenografts. Collectively, these findings suggest that YYJD exerts its anticancer activities through EGR1 activation, thus providing the evidence for its potential clinical application for lung cancer patients.

Keywords: ChIP-seq; EGR1; lung cancer; traditional Chinese medicine; transcriptome.

Figure 1

Yangyinjiedu (YYJD) induced anti‐tumour activities in lung cancer cells. A, The effects of various concentrations of YYJD on cellular proliferation of human lung cancer cell lines A549, NCI‐H2228, NCI‐H1299, NCI‐H1975, NCI‐HCC827, human normal bronchial epithelial cells (16HBE) and mouse Lewis lung carcinoma (LLC) were examined after 24, 48, and 72 h treatment respectively (*P < 0.05, ^# P < 0.001 compared with 16HBE group). B, YYJD induced cell cycle arrest at G₂/M phase. C, YYJD enhanced apoptosis rates of lung cancer cells in a concentration‐dependent manner. Data are shown as Mean ± SD from at least three independent experiments (*P < 0.05, **P < 0.01, ^# P < 0.001 compared with control group)

Figure 2

Characterization of the Yangyinjiedu (YYJD) induced differential gene expression in lung cancer cells. A, Scatter plot showed the differential gene expression pattern in A549 with YYJD treatment or with no YYJD treatment. Expression was shown as log10 of the FPKM+1, including up‐regulated (red) and down‐regulated (green) genes. B, Gene ontology analysis of the significantly differentially expressed genes in YYJD‐treated A549. C, Heatmap showed the 50 most up‐regulated and down‐regulated in YYJD‐treated A549. D, The alteration in early growth response 1 protein levels in YYJD‐treated lung cancer cell lines was examined by Western blot

Figure 3

Knockdown of early growth response 1 (EGR1) attenuated the Yangyinjiedu induced pro‐apoptosis effect in A549. A, The expression levels of EGR1 mRNA were detected by quantitative real‐time PCR. B, The expression levels of EGR1 protein were measured by Western blot. C, Cell viabilities were examined by CCK8 assay. D, Apoptotic cells were measured by flow cytometric assay. Ratios of early apoptosis and total apoptosis were analysed with flowjo software (**P < 0.01, ***P < 0.001 compared with control group)

Figure 4

Characterization of early growth response 1 (EGR1) directed transcriptional network in A549. A, A snapshot of the IGV genome browser showed the sequencing read signals of EGR1 binding sites. B, Genomic distribution of EGR1 binding sites across the Yangyinjiedu (YYJD) treated A549 genome. C, Venn diagram of differentially expressed genes and EGR1‐bound genes in YYJD‐treated A549. D, Gene ontology analysis of the up‐regulated EGR1 target genes (P < 0.05)

Figure 5

Tumour inhibitory effect of Yangyinjiedu in vivo. A, The tumour volumes were measured once every day. *P < 0.05, **P < 0.01 and ***P < 0.001. B, The comparison of tumour weights of four groups. *P < 0.05, **P < 0.01 and ***P < 0.001. C, Haematoxylin eosin staining of the tumour tissues (100× and 400× magnification). Representative images were shown from six mice in each group. D, The expression of early growth response 1 and KLF11 in tumour xenograft tissues was detected by immunohistochemistry (400×). Scale bars: 200 μm