## 3 Results
### Glycerol Sensitizes HCC827 GR Cells to Gefitinib  
The above data demonstrated that glycosylrol is able to inhibit MET activation both in vitro and in vivo. Given the critical role of elevated MET activation in the acquired resistance of cancer cells to gefitinib [8], we hypothesized that glycosylrol might potentiate gefitinib-resistant HCC827 GR cells to gefitinib. HCC827 GR cells were exposed to gefitinib alone or in combination with glycosylrol for 48 h, and the cytotoxicity was assessed by Annexin V/PI staining. As shown in Figure 3A, the cell death induction by the combination of gefitinib and glycosylrol was significantly stronger than by each agent alone. To critically determine the nature (additive or synergistic) of the enhanced cytotoxic effect by the combination, we calculated the combination index using the Chou-Talalay method [9]. The cells were treated with each agent alone and in combination in a fixed ratio of 1:1 for 48 h, and the changes of cell viability were measured by crystal violet staining. As shown in Figure 3B, gefitinib or glycosylrol treatment alone induced a concentration-dependent  
Figure 1: Exposure to glycosylrol (GC) leads to reduced viability of HCC827 GR cells in vitro. (**A**) Comparison of the sensitivity of HCC827 and HCC827 GR cells to gefitinib. (**B,C**) Comparison of MET activation between HCC827 and HCC827 GR cells (two replicates). (**D,E**) Effect of GC on cell viability of HCC827 GR cells. (**F,G**) Influence of GC on MET activation in HCC827 GR cells. * \(p<0.05\), **\(p<0.01\), *** \(p<0.001\), *** \(p<0.0001\). reduction of cell viability, whereas the combinations led to strongly further enhanced inhibitory effects. These data were then used to calculate the combination index using the median-effect equation of Chou-Talalay. As shown in Figure 3C, the values of the combination index were much less than 1 at all combinations tested, supporting a strongly synergistic effect induced by the combination of gefitinib and glycerol. Both the Benzofuranly Group at c-3/4 and the Isopentenyl Group at c-6 Are Indispensable for Glycerol to Exert the Sensitization Effect  
To decipher the structure-activity relationship of glycerol in terms of its sensitization effect, we assessed the combination effect of gefitinib and two analogues of glycerol, demethylsuberosin (De) and coumestrol (Coum). The chemical structures of glycerol, demethylsuberosin and coumestrol are shown in Figure 4A. These three compounds belong to the coumarin family, which is structurally constructed by a benzene ring fused to \(\alpha\)-pyrone ring. In comparison with glycerol, the benzofuranly group at c-3/4 is absent in demethylsuberosin, while the isopentenyl group at c-6 is absent in coumestrol. HCC827 GR cells were treated with gefitinib alone or in combination with demethylsuberosin or coumestrol at the same concentrations used with glycerol for 48 h, and the changes of cell viability  
Figure 3: Glycylrol sensitizes HCC827 GR cells to gefitinib. (**A**) Apoptosis induction by gefitinib alone or in combination with glycerol in HCC827 GR cells. (**B**) HCC827 GR cells were exposed to glycerol, gefitinib or their combination for 48 h, and the changes of cell viability were measured by crystal violet staining. (**C**) The combination index was calculated using Chou–Talalay equation based on the data generated above. *** \(p<0.01\), *** \(p<0.001\). Figure 2: Glycylrol inhibits tumor growth in HCC827 GR xenograft model. (**A**) Inhibitory effect of glycerol on tumor growth. (**B**) Reduction of the final tumor weight. (**C**) Bodyweight kinetics of mice. (**D,E**) Phospho-MET in tumor samples (each band represents one tumor sample). * \(p<0.05\), *** \(p<0.01\). are shown in Figure 4B,C. The synergistically inhibitory effect of the combination of gefitinib and glycosylrol was not observed by combining gefitinib with either demethylsuberosin or coumestrol. These results were consistent with the inability of these two analogues to inactivate MET (Figure 4D,E).