In this study, we have identified hydroxygenkwanin (HGK) as one of the active flavonoids that display anti-tumor activity against TKI-resistant NSCLC cells in vitro and in vivo. 2. NSCLC SYM2206 cells harboring EGFR mutations and xenograft mouse model to examine the antitumor activity of HGK on TKI-resistant NSCLC cells. The results showed that HGK suppressed cancer cell viability both in vitro and in vivo. Whole-transcriptome analysis suggests that EGFR is a potential upstream regulator that is involved in the gene expression changes affected by HGK. In support of this analysis, we presented evidence that HGK reduced the level of EGFR and inhibited several EGFR-downstream signalings. These results suggest that the antitumor activity of HGK against TKI-resistant NSCLC cells acts by enhancing the degradation of EGFR. Sieb.et Zucc. has been used in traditional Chinese medicine for thousands of years. The flower buds of this plant (Genkwa Flos) are mainly used for the treatment of cancer, asthma, and edema [6,7,8,9]. It contains several types of compounds, including flavonoids, biscoumarin, lignans, volatile oils, diterpene esters, chlorogenic acids, and phenolic glycosides. The flavonoids and diterpene esters are SYM2206 thought to be the major efficacy components [10,11]. Yuanhuadine, a Daphnane diterpene from Genkwa Flos, has been reported to inhibit the growth of human lung cancer cells, which was accompanied with cell cycle arrest, up-regulation of p21, and down-regulation of c-Myc, CDK2, CDK4, and cyclins [12]. Yuanhuadine also inhibits ligand-induced EGFR and c-Met signaling [12]. Yuanhuacine, a Daphnane diterpenoid from Genkwa Flos, has been shown to modulate the AMPK/mTORC2 signaling pathway and actin cytoskeleton organization in NSCLC cells [13]. The total flavonoids from Genkwa Flos have been shown to inhibit the growth of Lewis lung carcinoma in C57BL6 mice and colorectal cancer cells [14,15]. However, the active components in the flavonoids of Genkwa Flos have not been characterized. In this study, we have identified hydroxygenkwanin (HGK) as one of the active flavonoids that display anti-tumor activity against TKI-resistant NSCLC cells in vitro and in vivo. 2. Results 2.1. Isolation and Identification of Flavonoids from Genkwa Flos Genkwa Flos were extracted with methanol and then concentrated to give brown syrup. The syrup was partitioned first in CHCl3/water (1:1) and then in SYM2206 0.01; and *** 0.001, as analyzed with the unpaired DC displayed strong cytotoxicity against human lung cancer cells (NCI-H187) and a moderate toxicity against oral cavity cancer cells lines (KB) [19]. HGK induced DNA damage, cell cycle arrest, and cell apoptosis in glioma [20]. HGK inhibited cell migration, invasion, and proliferation in oral SYM2206 squamous cell carcinoma and hepatocellular carcinoma [21,22]. These data suggest that HGK is one of the active antitumor flavonoids in the Genkwa Flos extract. Table 1 Cytotoxic effects of the compounds Genkwanin, 3-Methoxy genkwanin, and hydroxygenkwanin (HGK). 0.05 and ** 0.01, as analyzed by unpaired = 5.5 10?22), oxidative phosphorylation (= 4.41 10?18), and protein ubiquitination pathway (= 5.23 10?16) (Table 2). Table 3 shows the top molecular and cellular functions of DE genes identified by IPA. The top five cellular functions that were affected by HGK suggest that the major activity of HGK is to affect the cell death and survival. Upstream regulator analysis in IPA was used to predict the upstream transcriptional regulators from the dataset. The overlap value was used to predict the potential transcriptional regulator through the gene expression database. EGFR (overlap = 5.23 10?16) (Table 2) based on transcriptome analysis. H1975 cells were treated cycloheximide in the presence or absence of HGK, and the levels of EGFR were determined by Western blot, to determine the effects of HGK on the protein stability of EGFR. As shown in Figure 3E, the amount of total EGFR low in the lack of HGK treatment gradually. However, the amount of EGFR was reduced in HGK-treated H1975 cells rapidly. We examined the result of proteasome inhibitor (MG132) over the EGFR balance in H1975 to handle if the instability of EGFR protein by HGK Tnfrsf1b treatment may be because of proteasome-mediated degradation [24,25]. The treating H1975 cells with MG132 at 10 M or lower concentrations acquired no influence on cell proliferation (Supplementary Amount S1A). In the current presence of MG132 at 10 M, the amount of EGFR was no reduced following longer.
