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

Hongchao Zhen,1 Guangxin Li,2 Pengfei Zhao,1 Ying Zhang,1 Jing Wang,1 Junxian Yu,3 Bangwei Cao1 1Cancer Center, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, People’s Republic of China; 2Radiation Oncology, Beijing Tsinghua Changgung Hospital, School of Clinical Medicine, Tsinghua University, Beijing 102218, People’s Republic of China; 3Department of Pharmacy, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, People’s Republic of ChinaCorrespondence: Bangwei CaoCancer Center, Beijing Friendship Hospital, Capital Medical University, #95 Yong an Road, Xicheng District, Beijing 100050, People’s Republic of ChinaTel/Fax +86-10-63139325Email oncology@ccmu.edu.cnObjective: 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: raltitrexed, apatinib, antitumor, ESCC, Akt, Erk