Effects of gefitinib on the proliferation of A549, H1975, PC9, and PC9/GR cells
To probe the sensitivity of different NSCLC cell lines to gefitinib, four NSCLC cell lines having a different EGFR status, including A549 (wild-type EGFR), H1975 (L858R and T790M mutation in EGFR exon 21-, PC9 (EGFR exon 19 deletion), and PC9/GR (gefitinib acquired resistant PC9 cells), were exposed to various concentrations of gefitinib (0, 0.625, 1.25, 2.5, 5, 10, 20 and 40 µmol/L) for 48 h. As shown in Fig. 1A, CCK-8 analysis disclosed that cell ability to PC9 cells significantlyl reduced in a concentration-dependent manner upon gefitinib treatment, whereas it had a minimal effect on cell viability at concentrations lower than 1.0 µmol/L. Moreover, cell viability was higher in the other gefitinib-resistant NSCLC cell lines (A549, H1975 and PC9/GR) than in PC9 cells. The IC50 values of the above-mentioned four NSCLC cells for gefitinib were calculated and shown in Fig. 1B and 1C, were 18.90 µmol/L, 16.40 µmol/L, 1.794 µmol/L and 15.99 µmol/L for A549, H1975, PC9, and PC9/GR, respectively (P < 0.05). Take together, these data indicated that the four NSCLC cell lines have different sensitivity to gefitinib, with PC9 cells being the most sensitive.
Upregulation of FGL1 correlates with gefitinib resistance
To explore the role of FGL1 in NSCLC cell resistance to gefitinib, we first measured FGL1 expression levels in normal bronchial epithelial cells BEAS-2B, and the four human NSCLC cell lines, A549, H1975, PC9, and PC9/GR, by western blot analysis and RT-qPCR. As shown in Fig. 2A, FGL1 expression was significantly higher in tumor cells (except A549 cells) than in normal BEAS-2B cells (P < 0.01), and among the four tumor cell lines, PC9 and PC9/GR cells exhibited higher FGL1 protein levels than A549 and H1975 cells (P < 0.05). The RT-qPCR results were consistent with the western blot data. These results suggested that FGL1 expression may relate to the mutation status of EGFR and contribute to gefitinib acquired resistance in NSCLC cells. To investigate whether FGL1 plays a role in the acquired resistance of NSCLC cells to EGFR-TKIs, we treated A549, H1975, PC9, and PC9/GR cells with gefitinib at various concentrations (0, 0.625 1.25, 2.5, 5, 10, 20, and 40 µmol/L). The results showed that gefitinib significantly increased FGL1 expression in PC9 and PC9/GR cells at both the mRNA and protein levels, in a concentration-dependent manner (P < 0.05) (Fig. 2B), whereas it had a minimal effect on FGL1 expression in A549 and H1975 cells (only mRNA expression increased in a concentration-dependent manner in H1975; P < 0.05). Together, these results indicated that the upregulation of FGL1 may be correlated with gefitinib acquired resistance in NSCLC cells with EGFR mutation.
Knockdown of FGL1 expression overcomes acquired resistance to gefitinib in PC9/GR cells
To investigate the effects of FGL1 on acquired resistance to gefitinib in NSCLC cells, we knocked down FGL1 in PC9/GR using siRNA. After siRNA transfection for 48 h, FGL1 expression was strongly reduced at both the mRNA and protein levels (Fig. 3A, P < 0.01). Next, we treated the FGL1-knockdown PC9/GR cells with gefitinib at different concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µmol/L) for 48 h. CCK-8 assays showed that downregulation of FGL1 significantly reduced cell viability and lowered the IC50 values (1.445 ± 0.617 µmol/L vs. 18.716 ± 2.167 µmol/L vs. 20.865 ± 3.164 µmol/L) in PC9/GR cells in response to gefitinib treatment as compared to non-transfected PC9/GR cells (Fig. 3B and 3C, P < 0.01). Flow cytometry (Fig. 3D) and colony-formation assays (Fig. 3E) revealed that FGL1 knockdown enhanced apoptosis and suppressed the colony number of PC9/GR cells, and even led to a considerable increase in gefitinib-induced apoptosis and a substantial decrease in colony number in PC9/GR cells treated with gefitinib (P < 0.05). These results suggested that knockdown of FGL1 increased the acquired resistance to gefitinib in PC9/GR cells, and overexpression of FGL1 contributed, at least in part, to apoptosis induced by gefitinib in PC9/GR cells in vitro.
Knockdown of FGL1 enhances the antitumor effects of gefitinib in vivo
To investigate the effects of FGL1 on the sensitivity of PC9/GR cells to gefitinib in vivo, a xenograft mouse model was established by subcutaneously injecting PC9/GR cells that had been transfected with lentivirus harboring NC-siRNA or FGL1-siRNA into BALB/c nude mice. When the average tumor volume reached 50 mm3, gefitinib was given daily at 30 mg/kg by oral gavage, after which tumor volume and body weight were monitored every other day. As shown in Fig. 4A, there was no significant difference in body weight between the groups (P > 0.05). However, tumor volumes (Fig. 4B, P < 0.05) and tumor weights (Fig. 4C, P < 0.05) were significantly lower in FGL1-siRNA-treated mice and in mice treated with FGL1-siRNA and gefitinib than in mice treated with NC-siRNA or gefitinib alone. Immunohistochemistry of the tumor tissues revealed that knockdown of FGL1 alone or co-culture with gefitinib significantly decreased the Ki-67 levels (Fig. 4D, P < 0.05). Flow-cytometric analysis of cells isolated from freshly isolated tumor tissues revealed that both FGL1 knockdown alone and FGL1 knockdown plus with gefitinib significantly increased the apoptotic rate (Fig. 4E, P < 0.05). Collectively, these results suggested that FGL1 knockdown suppresses tumor growth and significantly enhances the antitumor effect of gefitinib in vivo.
Knockdown of FGL1 overcomes acquired resistant to gefitinib via activating apoptotic pathway
To clarify the potential mechanisms by which FGL1 mediates gefitinib acquired resistance, we measured the expression of proteins related to apoptosis and proliferation in tumor cells by western blot analysis. PARP1 is a major member of the PARP family, and it plays an important role in DNA repair and cell death, proliferation, and differentiation as a sensor and signal transducer. Caspase 3 is the most important terminal shear enzyme involved in the process of cell apoptosis; tt recognizes the DEVD motif in the nuclear localization signal of its substrate PARP1 and disrupts the activity of the enzyme. EGFR is activated through phosphorylation. Therefore, we detected p-EGFR(1173) and p-EGFR(1068). As shown in Fig. 5A, knockdown of FGL1 expression in PC9/GR cells significantly decreased the protein levels of EGFR, p-EGFR(1173), p-EGFR(1068), whereas it decreased the levels of PARP1 and caspase 3, along with increased expression of cleaved PARP1 and cleaved caspase 3 (all P < 0.05). We detected the above-mentioned apoptosis-related proteins in tumor tissues freshly collected from mice inoculated with FGL1-siRNA-transfected PC9/GR cells and treated or not with gefitinib. As expected, gefitinib alone inhibited the expression of EGFR, p-EGFR (1173), and p-EGFR (1068), but did not affect the levels of PARP1 and caspase 3, whereas knockdown of FGL1 plus gefitinib treatment not only significantly decreased the protein levels of p-EGFR (Y1173 and Y1068), but also increased cleaved PARP1 and cleaved caspase 3 levels by cleaving PARP1 and caspase 3 (Fig. 5B, all P < 0.05). In summary, FGL1 depletion promotes the sensitivity of PC9/GR cells to gefitinib in vitro or in vivo, partly via activation of the PARP1/caspase 3 pathway.
Inhibition of FGL1 enhances the antitumor effects of gefitinib via inducing apoptosis in PC9 cells
To investigate whether FGL1 has the same apoptosis-promoting effect in gefitinib-sensitive NSCLC cells, we also knocked down FGL1 expression in PC9 cells using siRNA. As shown in Fig. 6A and 6B, FGL1 expression was obviously reduced in PC9 cells transfected with siRNA (P < 0.05). We found that FGL1 knockdown not only increased the sensitivity to gefitinib of PC9 cells, but also affected their survival in vitro. FGL1 knockdown significantly reduced cell proliferation (Fig. 6C) ,IC50 values for gefitinib and colony formation (Fig. 6E) and substantially increased the apoptotic rate (Fig. 6F) (all P < 0.05). Furthermore, FGL1 knockdown of significantly suppressed cell proliferation and lowered the IC50 value (0.143 ± 0.085 µmol/L vs. 1.092 ± 0.106 µmol/L) in PC9 cells exposed to gefitinib (Fig. 6C, P < 0.05). These data suggested that FGL1 depletion also promotes apoptosis and increases gefitinib sensitivity in PC9 cells. As for the mechanism, as shown in Fig. 6G, proteins related to apoptosis were expressed at the levels observed in PC9/GR cells after FGL1 knockdown in PC9 cells. Further, FGL1 knockdown inhibited EGFR and EGFR phosphorylation (p-EGFR1173 and p-EGFR1086), and decreased PARP1 and caspase 3 levels, regardless of the presence of gefitinib (P < 0.05), which implied that FGL1 promotes apoptosis of NSCLC cells PC9 through affecting the expression of PARP1/caspase 3 via lowering the expression and phosphorylation of EGFR.