## 4 Discussion  
Drug resistance is a major factor affecting the clinical efficacy of gefitinib. A MET-based mechanism is suggested to be an important contributor to the cancer cells refractory to gefitinib [10; 11]. HCC827, a human non--small-cell lung cancer (NSCLC) cell line harboring EGFR activating mutations, is initially sensitive to gefitinib. Long-term stepwise treatment with gefitinib leads to the cells' resistance to gefitinib (HCC827 GR). In the present study, we evaluated inhibitory effects of glycosylrol on HCC827 GR cells using both in vitro and in vivo models, and a possibility of glycosylrol as a sensitizer to improve the efficacy of gefitinib against its resistant cancer. Our data demonstrated that glycosylrol was able to inhibit the growth of HCC827 GR cells in cell culture systems and exerted a promising in vivo efficacy in the HCC827 GR xenograft mouse model. Moreover, a dramatically increased sensitivity of HCC827 GR cells to gefitinib was induced in response to glycerol. The findings  
Figure 4: Both the benzofuranyl group at c-3/4 and the isopentenyl group at c-6 are indispensable for glycosylrol to exert the sensitization effect. (**A**) Chemical structure of glycosylrol and its analogues, demethylsuberosin and coumestrol. (**B**) The changes of cell viability in response to gefitinib, demethylsuberosin, or their combination. (**C**) The changes of cell viability in response to gefitinib, coumestrol, or their combination. (**D**,**E**) Influence of glycosylrol and its analogues demethylsuberosin and coumestrol on MET activation. \({}^{*}p<0.05\). of the present study suggest that glycyrol holds promising potential to be developed as a novel agent to fight against gefitinib-resistant cancer. Proposed mechanisms that contributed to the resistance development of cancer cells to gefitinib mainly include mutation of EGFR-T790M, amplification of MET gene and its protein hyperactivation, amplification of ERBB2, transformation to small-cell lung cancer, and MET-mediated TOPK activation [8; 12; 13; 14; 15]. Our previous study showed that glycyrol was able to directly bind to and inactivate TOPK [7], which is supposed to contribute to the sensitization effect of glycyrol on HCC827 GR cells to gefitinib. In the present study, we addressed the role of MET inactivation in a glycyrol-mediated sensitization effect. Western blotting analysis of phospho-MET demonstrated that MET is highly activated in HCC827 GR cells, which is well in agreement with its gefitinib resistant feature, supporting a possible contribution of MET activation to HCC827 GR cell resistance to gefitinib. Targeting MET is therefore a reasonable approach to fight against the gefitinib-resistant cancer cells. Treatment with glycyrol effectively suppressed MET activation in cultured HCC827 GR cells, which provided a mechanistic explanation for the sensitization effect of glycyrol on HCC827 GR cells to gefitinib. Accordingly, the two analogues of glycyrol that lacked the ability to inactivate MET failed to potentiate HCC827 GR cells to gefitinib. Regarding the structure basis underlying the differentially suppressive effect on MET activation between glycyrol and its two analogues, our results revealed that both the benzofuranyl group at c-3/4 and the isopentenyl group at c-6 are necessary for glycyrol to exert an inhibitory effect on MET activation. These findings provided useful information for improving the anticancer activity of glycyrol through structure modification. Conceptualization, H.H. ; investigation, S.L. and S.Z. ; writing--original draft preparation, S.Z. ; writing--review and editing, H.H. and L.F.; supervision, H.H. All authors have read and agreed to the published version of the manuscript. This research was funded by the Ministry of Science and Technology of China [National Key Research and Development Program of China, 2018 YFC1603706]. Annal Care and procedures were approved by the Institutional Animal Care and Use Committee (Aw72101202-4-3, China Agricultural University). Not applicable.