Raltitrexed enhanced antitumor effect of anlotinib in human esophageal squamous carcinoma cells on proliferation, invasiveness, and apoptosis

Abstract

Background: Anlotinib is a multi-targeted receptor tyrosine kinase inhibitor (TKI) which has exhibited encouraging clinical activity in advanced non-small cell lung cancer (NSCLC) and soft tissue sarcoma. Raltitrexed is well known to be effective in the treatment of colorectal cancer in China. The present study aims to investigate the combinatory antitumor effect of anlotinib and raltitrexed on human esophageal squamous carcinoma cells and further explore the molecular mechanisms in vitro.

Methods: Human esophageal squamous cell lines KYSE-30 and TE-1 were treated with anlotinib or raltitrexed, or both, then cell proliferation was measured by MTS and colony formation assay; cell migration and invasion were detected by wound-healing and transwell assays; cell apoptosis rate was studied by flow cytometry and the transcription of apoptosis-associated proteins were monitored by quantitative polymerase chain reaction (qPCR) analysis. Finally, western blot was performed to check phosphorylation of apoptotic proteins after treatment.

Results: Treatment with raltitrexed and anlotinib showed enhanced inhibitory effects on cell proliferation, migration and invasiveness compared with raltitrexed or anlotinib monotherapy. Meanwhile, raltitrexed combined with anlotinib strongly increased cell apoptosis percentage. Moreover, the combined treatment down-regulated mRNA level of the anti-apoptotic protein Bcl-2 and invasiveness-associated protein matrix metalloproteinases-9 (MMP-9), while up-regulated pro-apoptotic Bax and caspase-3 transcription. Western blotting showed that the combination of raltitrexed and anlotinib could inhibit the expression of phosphorylated Akt (p-Akt), Erk (p-Erk) and MMP-9.

Conclusions: This study indicated that raltitrexed enhanced the antitumor effects of anlotinib on human ESCC cells by down-regulating phosphorylation of Akt and Erk, providing a novel treatment option for patients with esophageal squamous cell carcinoma (ESCC).

Keywords: Anlotinib; Antitumor; ESCC; P-Akt; P-Erk; Raltitrexed.

Fig. 1

Combinated effects of raltitrexed and anlotinib on cell proliferation in ESCC cells. A and B Anlotinib reduced cell proliferation rates of KYSE-30 (A) and TE-1 (B) cells in a dose-dependent manner after 24 h and 48 h treatment. C and D Raltitrexed reduced cell proliferation rates of KYSE-30 (C) and TE-1 (D) cells in a dose-dependent manner after 24 h and 48 h treatment. E and F Cell proliferation rates of KYSE-30 (E) and TE-1 (F) cells after treated with control, 20 μM anlotinib, 2.5 μM raltitrexed or 20 μM anlotinib + 2.5 μM raltitrexed for the indicated time in MTS assays. Data indicate means + SD of three biological replicates. Student’s t test; ^**P < 0.01, ^***P < 0.001 (versus control); ^#P < 0.05, ^###P < 0.001 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 2

Combinated effects of raltitrexed and anlotinib on viability of ESCC cells. A and C Colony formation ability of KYSE-30 (A) and TE-1 (C) cells inhibited by control, 20 μM anlotinib, 2.5 μM raltitrexed or 20 μM anlotinib + 2.5 μM raltitrexed. B and D Quantitative analysis of the number of colonies in KYSE-30 (B) and TE-1 (D) cells. Data indicate means + SD of three biological replicates. Student’s t test; ^***P < 0.001 (versus control); ^##P < 0.01 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 3

Combinated effects of raltitrexed and anlotinib on cell migration in ESCC cells. A and D KYSE-30 (A) and TE-1 (D) cells treated with control, 20 μM anlotinib, 2.5 μM raltitrexed or 20 μM anlotinib + 2.5 μM raltitrexed were scraped and imaged immediately (0 h) and later (24 h and 48 h), and images of the wound gap were taken. Scale bar = 100 μm. Quantitative analysis of the wound healing rate in KYSE-30 (B and C) and TE-1 (E and F) cells. Data indicate means + SD of three biological replicates. Student’s t test; ^***P < 0.001 (versus control); ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 4

Combinated effects of raltitrexed and anlotinib on cell invasion in ESCC cells. A and C After treatment with control, 20 μM anlotinib, 2.5 μM raltitrexed or 20 μM anlotinib + 2.5 μM raltitrexed for 24 h, invaded cells were stained and counted using microscopy in KYSE-30 (A) and TE-1 (C) cells. B and D Quantitative analysis of the number of invaded cells in KYSE-30 (B) and TE-1 (D) cells. Data indicate means + SD of three biological replicates. Student’s t test; ^***P < 0.001 (versus control); ^##P < 0.01, ^###P < 0.001 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 5

Combinated effects of raltitrexed and anlotinib on cell apoptosis in ESCC cells. A and C KYSE-30 (A) and TE-1 (C) cells were exposed to control, 20 μM anlotinib, 2.5 μM raltitrexed or 20 μM anlotinib + 2.5 μM raltitrexed for 48 h before Annexin V-FITC and 7AAD-PerCP staining and apoptosis percentage was detected by flow cytometry. B and D Quantitative analysis of the apoptotic rate in KYSE-30 (B) and TE-1 (D) cells. Q1 (necrosis), Q2 (late apoptosis), Q3 (early apoptosis) and Q4 (viable). Data indicate means + SD of three biological replicates. Student’s t test; ^***P < 0.01 (versus control); ^##P < 0.01 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 6

Combinated effect of raltitrexed and anlotinib on gene transcription in ESCC cells. mRNA levels of Bcl-2, MMP-9, Bax and Caspase-3 were measured by qPCR in either KYSE-30 (A-D) or TE-1 (E-H) cells after treated by 20 μM anlotinib, 2.5 μM raltitrexed, or both. Data indicate means + SD of three biological replicates. Student’s t test; ^*P < 0.05, ^**P < 0.01, ^***P < 0.01 (versus control); ^##P < 0.01, ^###P < 0.001 (versus 20 μM anlotinib or 2.5 μM raltitrexed)

Fig. 7

Protein expression after various treatments detected and quantified by Western blot analysis. KYSE-30 (A-H) and TE-1 (I-P) cells were treated with various treatments for 24 h. The protein levels of BAX, BCL-2, MMP-9, p-AKT, AKT, p-ERK, ERK, p-VEGFR-2, VEGFR-2, Caspase-3 and Cleaved Caspase-3 were determined by western blot analysis. β-ACTIN was detected as loading control. ^*P < 0.05, ^**P < 0.01 (versus control); ^#P < 0.05, ^##P < 0.01 (versus 20 μM anlotinib or 2.5 μM raltitrexed)