Raltitrexed Enhances the Antitumor Effect of Apatinib in Human Esophageal Squamous Carcinoma Cells via Akt and Erk Pathways

Abstract

Objective: Apatinib has been proved effective in the treatment of advanced gastric cancer and a variety of solid tumors. Raltitrexed is emerging as a promising alternative for treating advanced colorectal cancer in China. This work aims to study the combinatory antitumor effect of apatinib and raltitrexed on human esophageal squamous carcinoma cells (ESCC).

Materials and methods: Two VEGFR-2-positive human ESCC lines, KYSE-30 and TE-1, were treated with apatinib or raltitrexed, or both, then the cell proliferation rate was measured by MTS assay; cell migration and invasion were studied by transwell assays; cell apoptosis rate was determined by flow cytometry; cellular autophagy level affected was analyzed by Western blot analysis; finally, quantitative polymerase chain reaction (qPCR) was used to monitor transcription and Western blot was performed to check phosphorylation of apoptotic proteins after treatment.

Results: Both apatinib and raltitrexed significantly inhibited KYSE-30 and TE-1 cell proliferation in a dose-dependent manner. Treatment with both drugs showed enhanced inhibitory effects on cell proliferation, migration, and invasiveness compared with apatinib monotherapy. Apoptosis percentages in both cell lines were also remarkably increased by the combined treatment. Moreover, the combination of apatinib and raltitrexed down-regulated mRNA level of the anti-apoptotic protein Bcl-2, while up-regulated pro-apoptotic protein PARP, Bax, and caspase-3 transcription. Western blot analysis showed that phosphorylation levels of Erk, Akt, and invasiveness-associated protein matrix metalloproteinases-9 (MMP-9) were decreased in the combination group.

Conclusion: Taken together, these results indicate that raltitrexed enhances the antitumor effects of apatinib on human ESCC cells by down-regulating phosphorylation of Akt and Erk, implying a combination of raltitrexed and apatinib might be an effective option for treating esophageal squamous cell carcinoma patients.

Keywords: Akt; ESCC; Erk; antitumor; apatinib; raltitrexed.

Figure 1

Combined effects of raltitrexed and apatinib on cell proliferation in ESCC cells. (A) Detection of VEGFR2 expression in KYSE30 and TE-1 cells by Western blot. β-ACTIN was detected as loading control. (B and C) Cell proliferation rates of KYSE-30 (B) and TE-1 (C) cells after being treated with control, 50 µM apatinib, 2.5 µM raltitrexed, or 50 µM apatinib + 2.5 µM raltitrexed for the indicated time in MTS assays. Data indicate means+±SD of three biological replicates. Student’s t-test; *P<0.05, **P<0.01 (vs control); ^#P<0.05, ^##P<0.01 (vs 50 µM apatinib).

Figure 2

Combined effects of raltitrexed and apatinib on viability of ESCC cells. (A and C) Representative images of colony formation assay after KYSE-30 (A) and TE-1 (C) cells were treated with control, 50 µM apatinib, 2.5 µM raltitrexed, or 50 µM apatinib + 2.5 µM raltitrexed for 14 days. (B and D) Quantification of the colony formation assays in KYSE-30 (B) and TE-1 (D) cells shown in (A and C), respectively. Data indicate means±SD of three biological replicates. Student’s t-test; ***P<0.001 (vs control); ^###P<0.001 (vs 50 µM apatinib).

Figure 3

Combined effects of raltitrexed and apatinib on cell invasion in ESCC cells. Transwell analysis of KYSE-30 (A and B) and TE-1 (C and D) cells was performed 24 hours after treatment with control, 50 µM apatinib, 2.5 µM raltitrexed, or 50 µM apatinib + 2.5 µM raltitrexed. Data indicate means±SD of three biological replicates. Student’s t-test; *P<0.05, **P<0.01 (vs control); ^#P<0.05, ^##P<0.01 (vs 50 µM apatinib). Magnification ×100; Scale bar=200 µm.

Figure 4

Combined effects of raltitrexed and apatinib on cell apoptosis in ESCC cells. KYSE-30 (A and B) and TE-1 (C and D) cells were exposed to control, 50 µM apatinib, 2.5 µM raltitrexed, or 50 µM apatinib + 2.5 µM raltitrexed for 48 hours before Annexin V-FITC and PI staining and apoptosis percentage was detected by flow cytometry. Data indicate means±SD of three biological replicates. Student’s t-test; *P<0.05, **P<0.01 (vs control); ^#P<0.05, ^##P<0.01 (vs 50 µM apatinib).

Figure 5

Combined effect of raltitrexed and apatinib on apoptosis-associated gene transcription in ESCC cells. mRNA levels of PARP, Bax, Caspase-3, and Bcl-2 were measured by qPCR in either KYSE-30 (A–D) or TE-1 (E–H) cells after treated by 50 µM apatinib, 2.5 µM raltitrexed, or both. Data indicate means±SD of three biological replicates. Student’s t-test; *P<0.05, **P<0.01 (vs control); ^#P<0.05, ^##P<0.01 (vs 50 µM apatinib).

Figure 6

Protein expression after various treatments detected and quantified by Western blot. (A)KYSE-30 cells were treated by 50 µM apatinib, or 2.5 µM raltitrexed, or both for 48 hours before expression of MMP-9, BCL-2, BAX, p-ERK, ERK, p-AKT, AKT, p-VEGFR2, VEGFR2, Caspase-3, Cleaved Caspase-3, and LC3 were examined by Western blot analysis. β-ACTIN was detected as loading control. (B-I) Quantification of different protein expression levels. **P<0.01 (vs control); ^##P<0.01 (vs 50 µM apatinib).

Figure 7

Protein expression after various treatments detected and quantified by Western blot. (A)TE-1 cells were treated by 50 µM apatinib, 2.5 µM raltitrexed, or both for 48 hours before expression of MMP-9, BCL-2, BAX, p-ERK, ERK, p-AKT, AKT, p-VEGFR2, VEGFR2, Caspase-3, Cleaved Caspase-3, and LC3 were examined by Western blot analysis. β-ACTIN was detected as loading control. (B-I) Quantification of different protein expression levels. **P<0.01 (vs control); ^##P<0.01 (vs 50 µM apatinib).