Chloroquine enhances gefitinib cytotoxicity in gefitinib-resistant nonsmall cell lung cancer cells

Abstract

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib, are effective for non-small cell lung cancer (NSCLC) patients with EGFR mutations. However, these patients eventually develop resistance to EGFR-TKI. The goal of the present study was to investigate the involvement of autophagy in gefitinib resistance. We developed gefitinib-resistant cells (PC-9/gef) from PC-9 cells (containing exon 19 deletion EGFR) after long-term exposure in gefitinib. PC-9/gef cells (B4 and E3) were 200-fold more resistant to gefitinib than PC-9/wt cells. Compared with PC-9/wt cells, both PC-9/gefB4 and PC-9/gefE3 cells demonstrated higher basal LC3-II levels which were inhibited by 3-methyladenine (3-MA, an autophagy inhibitor) and potentiated by chloroquine (CQ, an inhibitor of autophagolysosomes formation), indicating elevated autophagy in PC-9/gef cells. 3-MA and CQ concentration-dependently inhibited cell survival of both PC-9wt and PC-9/gef cells, suggesting that autophagy may be pro-survival. Furthermore, gefitinib increased LC3-II levels and autolysosome formation in both PC-9/wt cells and PC-9/gef cells. In PC-9/wt cells, CQ potentiated the cytotoxicity by low gefitinib (3 nM). Moreover, CQ overcame the acquired gefitinib resistance in PC-9/gef cells by enhancing gefitinib-induced cytotoxicity, activation of caspase 3 and poly (ADP-ribose) polymerase cleavage. Using an in vivo model xenografting with PC-9/wt and PC-9/gefB4 cells, oral administration of gefitinib (50 mg/kg) completely inhibited the tumor growth of PC-9/wt but not PC-9/gefB4cells. Combination of CQ (75 mg/kg, i.p.) and gefitinib was more effective than gefitinib alone in reducing the tumor growth of PC-9/gefB4. Our data suggest that inhibition of autophagy may be a therapeutic strategy to overcome acquired resistance of gefitinib in EGFR mutation NSCLC patients.

Fig 1. Basal autophagy levels in PC-9/wt and PC-9/gef cells.

(A) Representative Western blot data showed the basal LC3-II levels in PC-9/wt and PC-9/gef cells (B4 and E3). (B) Immunofluorescent staining studies were performed using LC3 antibody to show LC3 puncta in the PC-9/wt and PC-9/gefB4 cells. (C and D): PC-9/wt, PC-9/gefB4 and PC-9/ gefE3 cells were treated with 3-methyladenine (3-MA, 10 mM) and chloroquine (CQ, 1 and 10 μM) for 24 h. Total protein of treated cells was harvested; LC3-I and II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments.

Fig 2. Effects of autophagy inhibitors on proliferation of PC-9/wt and PC-9/gef cells.

PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with (A) 3-MA (0.1–1 mM) and (B) CQ (5–15 μM) for 96 h. Cell viability was determined using SRB assay. Values are the mean ± S.E.M. (n = 3). *P < 0.05 statistically significant in the 3-MA or CQ-treated groups compared with the controls.

Fig 3. Effects of gefitinib on LC3-II level in PC-9/wt and PC-9/gef cells.

(A) PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with gefitinib (nM) at various concentrations for 24 h. Total protein of treated cells was harvested; LC3-I and II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments. (B) PC-9/wt and PC-9/gefB4 cells were treated with gefitinib (1 μM) for 24 h. Immunofluorescent staining and fluorescent staining studies were performed using LC3 antibody and LysoTracker red (LysoTR) respectively to show puncta in the cells. Green, FITC-labeled LC3; red, LysoTR; blue, DAPI-labeled nucleus.

Fig 4. Autophagy activation and cytotoxicity by gefitinib and chloroquine in PC-9/wt and PC-9/gefB4 cells.

(A) PC-9/wt and PC-9/gefB4 were treated with gefitinib (100 nM) and chloroquine (CQ, 5, 10 μM) for 24 h. Total protein of treated cells was harvested. LC3-II levels were analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments. (B) PC-9/wt and (C) PC-9/gefB4 cells were cultured with gefitinib (100 nM) and CQ (5, 10 μM) for 96 h. (D) PC-9/wt cells were cultured with gefitinib (3 nM) and CQ (5, 10 μM) for 96 h. Cell viability was determined using SRB assay. Values are the mean ± S.E.M. (n = 3). *P < 0.05 statistically significant in the gefitinib or CQ-treated groups compared with the controls; # P< 0.05 statistically significant in gefitinib plus CQ-treated groups compared with gefitinib only groups.

Fig 5. Activation of apoptotic pathway by gefitinib and chloroquine in PC-9/wt and PC-9/gef ells.

PC-9/wt, PC-9/gefB4 and PC-9/gefE3 cells were treated with gefitinib (100 nM) and chloroquine (CQ, 10 μM) for 24 h. Total protein of treated cells was harvested. Procaspase 3, active caspase 3, PARP cleavage levels was analyzed with Western blot assay. Each lane contained 30 μg protein for all experiments. Results were repeated in independent experiments.

Fig 6. The anti-tumor effect of gefitinib and chloroquine in the mouse xenograft model.

Balb/c nude mice bearing PC-9/wt (A) and PC-9/gefB4 (B) xenografts were treated with vehicles as control (○, n = 4 for PC-9/wt and PC-9/gefB4, respectively), gefitinib (50 mg/kg/day by a gavage,◆; n = 5 for PC-9/wt and PC-9/gefB4, respectively), chloroquine (CQ, 75 mg/kg, i.p. ▲; n = 4 for PC-9/wt and PC-9/gefB4, respectively), or a combination of both (■; n = 5 for PC-9/wt and PC-9/gefB4, respectively).Tumors were allowed to grow to 200 mm³ before drug treatments. Values are the mean ± S.E.M. (n = 4–5). ** p<0.001, statistically significant in gefitinib and gefitinib plus CQ groups compared with the vehicle group in PC-9/wt tumor xenografts. * p<0.05, statistically significant in gefitinib plus CQ group compared with the vehicle group; # p<0.05 statistically significant in gefitinib plus CQ group compared with gefitinib alone in PC-9/gefB4 tumor xenografts by Independent-Samples T Test. (C) Representative data show 4 mice with PC-9/wt xenografts and 4 mice with PC-9/gefB4 xenografts which were treated with vehicle, CQ only, gefitinib only and gefitinib plus CQ.