Ate the inhibition of xenograft growth by quercetin and brigatinib, the levels of AXL, phospho (p)STAT3, phospho (p)EGFR, and cleaved caspase three were examined in the excised tumor xenografts. As shown in Figure 4E, the levels of AXL and pSTAT3 but not pEGFR had been reduced in quercetintreated tumors. In contrast, the amount of pEGFR was reduced in brigatinibtreated tumors, while the levels of AXL and pSTAT3 were not reduced. The cleavage of caspase three was improved in tumor xenografts treated with either quercetin or brigatinib and was considerably elevated in tumors treated with each quercetin and brigatinib. These final results are constant with the model in which quercetin inhibits tumor growth by suppressing the expression of AXL and activation of STAT3 in NSCLC cells, though brigatinib inhibits tumor growth by suppressing pEGFR in NSCLC cells (Figure 4F). four. Discussion Quercetin was demonstrated to exert antitumor activity in NSCLCs harboring wildtype EGFR [15,29]. Within this study, we demonstrated that quercetin exhibited a greater cytotoxic impact in NSCLC cells harboring TKIresistant EGFR mutations than wildtype EGFR (Figure 1). The higher cytotoxic effects of quercetin observed in TKIresistant cells appeared to correlate using the extent of apoptosis induction, as indicated by the detection of higher PARP cleavage in TKIresistant cells (Figure 2A). As quercetin was shown to induce autophagy to market apoptosis in A549 cells , we examined whether or not apoptosis induction might be associated with autophagy induction. Consistent having a earlier study, we observed that quercetin increased the expression of LC3II, an autophagy marker, in quercetintreated A549 cells (Figure 2A). Nevertheless, a rise in LC3II was not detected in quercetintreated NSCLC cells harboring activating EGFR mutations. These results suggest that quercetininduced apoptosis in NSCLC cells harboring activating EGFR mutations is independent of autophagy induction. Quercetin was reported to induce apoptosis by inhibiting the AXLSTAT3 axis pathway in glioblastoma cells . Since the acquisition on the EGFRC797S mutation resulted inside the upregulation of AXL  and cells harboring C797S (H1975MS35) had been extra sensitive to quercetin than the parental H1975 cells (Figures 1 and 2), we hypothesized that apoptosis induction by quercetin in H1975MS35 cells may perhaps be related to the inhibition of AXL. As shown in Figure 3A, quercetin downregulated the expression of AXL in each H1975 and H1975MS35 cells. As shown in Figure 3C,D, the inhibition of AXL occurred at both the transcriptional and posttranscriptional levels. Hypoxiainducible factor 1 (HIF1) and activator protein 1 (AP1) are known to be involved in modulating the transcriptional expression of AXL . As quercetin is actually a potent BSc5371 In Vivo inhibitor of AP1 and HIF1 , it truly is probably that quercetin might impact AXL transcription by inhibiting AP1 and HIF1. To understand how quercetin may possibly affect the posttranslational decreases in AXL, we performed a molecular Monocaprylin Autophagy docking evaluation. Quercetin was docked into the crystal structure on the AXL protein (PDB ID: 5U6B), and the docking analysis final results have been visualized with Learn Studio. The docking analysis showed docking potential with the kinase domain of AXL (Supplementary Figure S3A). Based on the bonding distance, bonding type, plus the position of quercetin, Lys567, Pro621, Arg676, and Asp690 of AXL could be imporBiomolecules 2021, 11,10 oftant binding web sites for forming hydrogen bonds with quercetin (Supplement.