Schedule-dependent cytotoxic synergism of pemetrexed and erlotinib in BXPC-3 and PANC-1 human pancreatic cancer cells

Abstract

Previous studies have shown that both pemetrexed, a cytotoxic drug, and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), inhibit the cell growth of pancreatic cancer cells. However, whether they exert a synergistic antitumor effect on pancreatic cancer cells remains unknown. The present study aimed to assess the synergistic effect of erlotinib in combination with pemetrexed using different sequential administration schedules on the proliferation of human pancreatic cancer BXPC-3 and PANC-1 cells and to probe its cellular mechanism. The EGFR and K-ras gene mutation status was examined by quantitative PCR high-resolution melting (qPCR-HRM) analysis. BXPC-3 and PANC-1 cells were incubated with pemetrexed and erlotinib using different administration schedules. MTT assay was used to determine cytotoxicity, and cell cycle distribution was determined by flow cytometry. The expression and phosphorylation of EGFR, HER3, AKT and MET were determined using Western blotting. Both pemetrexed and erlotinib inhibited the proliferation of BXPC-3 and PANC-1 cells in a dose- and time-dependent manner in vitro. Synergistic effects on cell proliferation were observed when pemetrexed was used in combination with erlotinib. The degree of the synergistic effects depended on the administration sequence, which was most obvious when erlotinib was sequentially administered at 24-h interval following pemetrexed. Cell cycle studies revealed that pemetrexed induced S arrest and erlotinib induced G(0)/G(1) arrest. The sequential administration of erlotinib following pemetrexed induced S arrest. Western blot analyses showed that pemetrexed increased and erlotinib decreased the phosphorylation of EGFR, HER3 and AKT, respectively. However, both pemetrexed and erlotinib exerted no significant effects on the phosphorylation of c-MET. The phosphorylation of EGFR, HER3 and AKT was significantly suppressed by scheduled incubation with pemetrexed followed by erlotinib, but not by concomitant or sequential incubation with erlotinib followed by pemetrexed. In summary, our results demonstrated that the combined use of erlotinib and pemetrexed exhibited a strong synergism in BXPC-3 and PANC-1 cells. The inhibitory effects were strongest after sequential administration of pemetrexed followed by erlotinib. The synergistic effects may be related to activation of the EGFR/HER3/AKT pathway induced by pemetrexed.

Figure 1.

Effects of erlotinib and pemetrexed on the cell proliferation of BXPC-3 and PANC-1 cells. (A) Erlotinib and pemetrexed inhibited cell proliferation in a dose-dependent manner when they were added to quiescent BXPC-3 cells. (B) Erlotinib and pemetrexed inhibited cell proliferation in a dose-dependent manner when they were added to quiescent PANC-1 cells. (C) Time course of erlotinib and pemetrexed-induced cell proliferation in BXPC-3 cells. (D) Time course of erlotinib and pemetrexed-induced cell proliferation in PANC-1 cells. The curves represented cell survival rate in the presence of different concentrations of erlotinib and pemetrexed. Cells in 96-well plates were rendered quiescent in RPMI-1640 medium. Quiescent monolayers were stimulated with erlotinib or pemetrexed at the indicated concentrations and then cultured for a further 72 h. ⋄ and ♦ represent the BXPC-3 and PANC-1 cells, respectively. Data are reported as the means ± SE; n=12.

Figure 2.

Effects of different exposure schedules of erlotinib and pemetrexed on cell survival in vitro measured by means of the MTT colorimetric assay. (A) The five exposure schedules tested: i) erlotinib treatment alone (E group); ii) pemetrexed treatment alone (P group); iii) concurrent treatment of erlotinib and pemetrexed for 72 h (E+P group); iv) 24-h exposure of erlotinib followed by pemetrexed for a total of 72 h (E→P group); v) 24-h exposure of pemetrexed followed by erlotinib for a total of 72 h (P→E group). (B) Effect of different administration schedules of erlotinib and pemetrexed on the proliferation of BXPC-3 cells. (C) Effect of different administration schedules of erlotinib and pemetrexed on the proliferation of PANC-1 cells. Data are reported as the means ± SE; n=6. ^*P<0.05 vs. control; ^#P<0.05 vs. E and P groups; ^▵P<0.05 vs. P→E group.

Figure 3.

Alterations is the cell cycle upon different exposure schedules of pemetrexed and erlotinib in BXPC-3 cells. BXPC-3 cells were treated with different schedules of pemetrexed (39.86 μmol/l) and/or erlotinib (8.86 μmol/l) alone or in combination. The exposure schedules tested and the concentrations used in the cell cycle distribution were consistent with their effects on cell growth. Columns, the mean of three independent experiments; bars, SD.

Figure 4.

Effect of different exposure schedules of erlotinib and pemetrexed on EGFR/HER3/AKT signaling pathways and cMET in BXPC-3 cells. Western blotting indicates the expression and phosphorylation of EGFR, HER3, AKT and MET in BXPC-3 cells. Cells were treated as described in Fig. 2A.

Figure 5.

Effect of different exposure schedules of erlotinib and pemetrexed on EGFR/HER3/AKT signaling pathways and cMET in PANC-1 cells. Western blotting indicates the expression and phosphorylation of EGFR, HER3, AKT and MET in PANC-1 cells. Cells were treated as described in Fig. 2A.