Favorable effect of the combination of vinorelbine and dihydropyrimidine dehydrogenase‑inhibitory fluoropyrimidine in EGFR‑mutated lung adenocarcinoma: retrospective and in vitro studies

Abstract

Although cytotoxic chemotherapy is essential in epidermal growth factor receptor (EGFR)‑mutated non‑small cell lung cancer (NSCLC), it is unclear which regimen is most effective. We retrospectively compared the efficacy of standard platinum‑based chemotherapy with that of combination chemotherapy using vinorelbine (VNR) plus dihydropyrimidine dehydrogenase‑inhibitory fluoropyrimidine (DIF) in EGFR‑mutated lung adenocarcinomas, and we investigated a potential mechanism by which the combination chemotherapy of VNR + DIF was favorable in the treatment of EGFR‑mutated lung adenocarcinoma in vitro. In our retrospective analysis, the response rate and disease control rate afforded by the VNR + DIF treatment tended to be better than those by platinum‑based chemotherapy, and the progression‑free survival of the 24 VNR + DIF‑treated patients was significantly longer than that of the 15 platinum‑based chemotherapy patients. In EGFR‑mutated PC9 cells, VNR induced EGFR dephosphorylation at a clinically achievable concentration. 1BR3‑LR cells, a line of fibroblast cells transfected with a mutant EGFR construct, were completely resistant to gefitinib in the medium containing 10% fetal bovine serum (FBS), whereas the sensitivity of these cells to gefitinib was increased in 0.5% FBS‑containing medium. Similarly, the sensitivity of 1BR3‑LR cells to VNR was increased when they were cultured in low‑serum condition. In addition, sodium orthovanadate (Na3VO4) inhibited the EGFR dephosphorylation induced by VNR or gefitinib and suppressed the cell growth inhibition by these agents in PC9 cells. VNR and gefitinib showed synergistic cell growth inhibition in combination with 5‑fluorouracil (5‑FU) in PC9 cells. We propose that the EGFR dephosphorylation induced by VNR is related to cell growth inhibitory activity of VNR, and that this is one of the mechanisms of the synergistic effect of VNR + 5‑FU in EGFR‑mutated lung cancer cells. In conclusion, the combination chemotherapy of VNR + DIF may be a promising treatment for NSCLC patients with EGFR mutations.

Figure 1

Kaplan-Meier curves of the progression-free survival (PFS) of the patients who received vinorelbine (VNR) + dehydrogenase-inhibitory fluoropyrimidine (DIF) chemotherapy (n=24) or platinum-based chemotherapy (n=15).

Figure 2

Sensitivity of PC9 cells to vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU). (A–C) PC9 cells were treated with the indicated concentrations of VNR, CDDP, or 5-FU for 24, 24 and 72 h, respectively. The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of untreated cells using a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 72 h after the start of drug exposure. Data are means ± SD of three separate experiments.

Figure 2

Sensitivity of PC9 cells to vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU). (A–C) PC9 cells were treated with the indicated concentrations of VNR, CDDP, or 5-FU for 24, 24 and 72 h, respectively. The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of untreated cells using a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 72 h after the start of drug exposure. Data are means ± SD of three separate experiments.

Figure 2

Sensitivity of PC9 cells to vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU). (A–C) PC9 cells were treated with the indicated concentrations of VNR, CDDP, or 5-FU for 24, 24 and 72 h, respectively. The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of untreated cells using a 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay 72 h after the start of drug exposure. Data are means ± SD of three separate experiments.

Figure 3

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation in PC9 cells. (A) PC9 cells were treated with the indicated concentrations of VNR for 24 h (upper panels), or 20 nM VNR for the indicated time (lower panels). Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B and C) PC9 cells were treated with the indicated concentrations of CDDP or 5-FU for 24 h and processed as described above.

Figure 3

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation in PC9 cells. (A) PC9 cells were treated with the indicated concentrations of VNR for 24 h (upper panels), or 20 nM VNR for the indicated time (lower panels). Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B and C) PC9 cells were treated with the indicated concentrations of CDDP or 5-FU for 24 h and processed as described above.

Figure 3

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation in PC9 cells. (A) PC9 cells were treated with the indicated concentrations of VNR for 24 h (upper panels), or 20 nM VNR for the indicated time (lower panels). Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B and C) PC9 cells were treated with the indicated concentrations of CDDP or 5-FU for 24 h and processed as described above.

Figure 4

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation and cell growth inhibition in 1BR3-LR cells. (A) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B–E) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 72 h in the medium containing 10% (solid line) or 0.5% (dotted line) fetal bovine serum (FBS). The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as means ± SD of three separate experiments.

Figure 4

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation and cell growth inhibition in 1BR3-LR cells. (A) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B–E) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 72 h in the medium containing 10% (solid line) or 0.5% (dotted line) fetal bovine serum (FBS). The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as means ± SD of three separate experiments.

Figure 4

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation and cell growth inhibition in 1BR3-LR cells. (A) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B–E) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 72 h in the medium containing 10% (solid line) or 0.5% (dotted line) fetal bovine serum (FBS). The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as means ± SD of three separate experiments.

Figure 4

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation and cell growth inhibition in 1BR3-LR cells. (A) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B–E) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 72 h in the medium containing 10% (solid line) or 0.5% (dotted line) fetal bovine serum (FBS). The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as means ± SD of three separate experiments.

Figure 4

Effects of vinorelbine (VNR), cisplatin (CDDP), and 5-fluorouracil (5-FU) on epidermal growth factor receptor (EGFR) phosphorylation and cell growth inhibition in 1BR3-LR cells. (A) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (B–E) 1BR3-LR cells were treated with the indicated concentrations of gefitinib, VNR, CDDP, or 5-FU for 72 h in the medium containing 10% (solid line) or 0.5% (dotted line) fetal bovine serum (FBS). The survival cell fraction is expressed as the percentage of optical density (% OD) in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are presented as means ± SD of three separate experiments.

Figure 5

Effects of sodium orthovanadate (Na₃VO₄) on epidermal growth factor receptor (EGFR) phosphorylation and the cell growth inhibition by gefitinib or vinorelbine (VNR) in PC9 cells. (A and B) PC9 cells were treated with 50 nM gefitinib or 20 nM VNR in the presence or absence of 50 μM Na₃VO₄ for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (C and D) PC9 cells were treated with the indicated concentrations of gefitinib or VNR in the presence or absence of 50 μM Na₃VO₄ for 72 h. The survival cell fraction is expressed as the % OD in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are means ± SD of three separate experiments.

Figure 5

Effects of sodium orthovanadate (Na₃VO₄) on epidermal growth factor receptor (EGFR) phosphorylation and the cell growth inhibition by gefitinib or vinorelbine (VNR) in PC9 cells. (A and B) PC9 cells were treated with 50 nM gefitinib or 20 nM VNR in the presence or absence of 50 μM Na₃VO₄ for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (C and D) PC9 cells were treated with the indicated concentrations of gefitinib or VNR in the presence or absence of 50 μM Na₃VO₄ for 72 h. The survival cell fraction is expressed as the % OD in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are means ± SD of three separate experiments.

Figure 5

Effects of sodium orthovanadate (Na₃VO₄) on epidermal growth factor receptor (EGFR) phosphorylation and the cell growth inhibition by gefitinib or vinorelbine (VNR) in PC9 cells. (A and B) PC9 cells were treated with 50 nM gefitinib or 20 nM VNR in the presence or absence of 50 μM Na₃VO₄ for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (C and D) PC9 cells were treated with the indicated concentrations of gefitinib or VNR in the presence or absence of 50 μM Na₃VO₄ for 72 h. The survival cell fraction is expressed as the % OD in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are means ± SD of three separate experiments.

Figure 5

Effects of sodium orthovanadate (Na₃VO₄) on epidermal growth factor receptor (EGFR) phosphorylation and the cell growth inhibition by gefitinib or vinorelbine (VNR) in PC9 cells. (A and B) PC9 cells were treated with 50 nM gefitinib or 20 nM VNR in the presence or absence of 50 μM Na₃VO₄ for 24 h. Total cellular protein (1 mg) from cell lysate was immunoprecipitated using anti-EGFR antibody and subjected to a western blot analysis with anti-phosphotyrosine (p-EGFR, upper panel), and the membrane was stripped of bound antibodies and re-probed with anti-EGFR antibody (middle panel). Total cellular protein (20 μg) of the same lysate was subjected to a western blot analysis with β-actin (lower panel). (C and D) PC9 cells were treated with the indicated concentrations of gefitinib or VNR in the presence or absence of 50 μM Na₃VO₄ for 72 h. The survival cell fraction is expressed as the % OD in reference to the OD of the untreated cells in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Data are means ± SD of three separate experiments.

Figure 6

The synergistic cell growth inhibition by the combination of gefitinib or vinorelbine (VNR) with 5-fluorouracil (5-FU) in PC9 cells. (A) PC9 cells were treated with either a single agent or the simultaneous combination of 5-FU and gefitinib for 72 h. (B) PC9 cells were treated with either a single agent or the sequential combination of VNR for 24 h and 5-FU for the next 72 h. The viabilities of the cells were determined in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) for each concentration of 5-FU was calculated by the Chou-Talalay method.

Figure 6

The synergistic cell growth inhibition by the combination of gefitinib or vinorelbine (VNR) with 5-fluorouracil (5-FU) in PC9 cells. (A) PC9 cells were treated with either a single agent or the simultaneous combination of 5-FU and gefitinib for 72 h. (B) PC9 cells were treated with either a single agent or the sequential combination of VNR for 24 h and 5-FU for the next 72 h. The viabilities of the cells were determined in an 3-(4,5-dimethylhiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The combination index (CI) for each concentration of 5-FU was calculated by the Chou-Talalay method.