Dihydroartemisinin inhibits the migration of esophageal cancer cells by inducing autophagy

Abstract

Esophageal cancer (EC) is a complex gastrointestinal malignancy and its global incidence rate ranks 7th among all cancer types. Due to its aggressive nature and the potential for early metastasis, the survival rates of patients with EC are poor. Dihydroartemisinin (DHA) is the primary active derivative of artemisinin, and, as well as its use as an anti-malarial, DHA has also exhibited antitumor activity in various cancer models, such as cholangiocarcinoma, head and neck carcinoma, and hepatocellular carcinoma cells. However, the molecular mechanisms underlying the antitumor effect of DHA in the treatment of EC remains poorly understood. The results of the present study demonstrated that DHA significantly inhibited the migration of TE-1 and Eca-109 EC cells in a dose-dependent manner by activating autophagy. DHA treatment also significantly reversed epithelial-mesenchymal transition (EMT) by downregulating the EMT-associated markers, N-cadherin and vimentin, and upregulating the expression of E-cadherin. Mechanistically, DHA treatment decreased Akt phosphorylation and inhibited the Akt/mTOR signaling pathway, leading to the activation of autophagy. The levels of the autophagy-associated proteins were suppressed and DHA-mediated inhibition of migration in EC cells was reversed when an active form of Akt was overexpressed. In conclusion, the present study demonstrated the potential value of DHA in the treatment of EC, and revealed the underlying mechanism by which FDHA inhibits cellular migration.

Keywords: Akt; autophagy; dihydroartemisinin; esophageal cancer; migration.

Figure 1.

DHA inhibits esophageal cancer cell migration in a concentration-dependent manner. (A) A total of 1×10⁶Eca-109 cells were seeded into 6-well plates and incubated overnight, following which the monolayers were scratched with 1-ml tips and the cells were cultured with media containing 3% FBS, and treated with 1, 2 or 5 µg/ml DHA for 24 h. Images of wound closure distances were captured 0 and 24 h post-treatment. Scale bar, 100 µm. (B) 2×10⁴ Eca-109 cells were inoculated into Transwell chambers with serum-free medium, treated with 2 µg/ml DHA for 24 h, and then fixed and stained. Migration ability was measured using the mean number of cells in 20 visual fields. Scale bar, 50 µm. (C) A total of 1×10⁶ TE-1 cells were seeded into 6-well plates and incubated overnight, the description of wound healing assay was similar as above. Scale bar, 100 µm. (D) 2×10⁴ TE-1 cells were inoculated into Transwell chambers with serum-free medium and performed as above. Migration ability was measured using the mean number of cells in 20 visual fields. Scale bar, 50 µm. Data are represented as the mean ± standard deviation from 3 independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. control. DHA, dihydroartemisinin.

Figure 2.

DHA induces autophagy in TE-1 and Eca-109 cells. (A) A total of 1×10⁵ cells were seeded into a 24-well plates and incubated overnight, and then transfected with an GFP-LC3 expression plasmid for 6 h. The cells were treated with 2 µg/ml DHA for 24 h, following which the cells were fixed and stained with DAPI. LC3 expression was analyzed using fluorescence microscopy, and the number of GFP-LC3 puncta per cell was determined using Image J. Yellow arrows indicate LC3 protein. Scale bar, 5 µm. A total of 1×10⁶ (B) Eca-109 and (C) TE-1 cells were seeded into 6-well plates and incubated overnight, and then treated with 2 µg/ml DHA for 24 h. Levels of autophagy-associated proteins were detected using western blot analysis, and the relative density was determined using ImageJ software. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 and **P<0.01 vs. respective control. DHA, dihydroartemisinin; GFP, green fluorescent protein; LC3, microtubule-associated protein 1A/1B-light chain 3; SQSTM, sequestosome 1; ns, not significant.

Figure 3.

DHA inhibits EC cell migration by activating autophagy. (A) A total of 2×10⁴ EC cells were inoculated into Transwell chambers with serum-free medium and treated with 2 µg/ml DHA, 2 mmol/l 3-MA or a combination of both for 24 h. Membranes were then fixed and stained. Migration was recorded as the mean number of cells in 20 visual fields. Scale bar, 50 µm. The levels of autophagy-associated proteins in (B) Eca-109 and (C) TE-1 cells were detected using western blot analysis, and the relative density was determined using ImageJ software. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 and **P<0.01. DHA, dihydroartemisinin; EC, esophageal cancer; 3-MA, 3-methyladenine; LC3, microtubule-associated protein 1A/1B-light chain 3; SQSTM, sequestosome 1.

Figure 4.

DHA induces autophagy via the Akt-mTOR signaling pathway. A total of 1×10⁶ (A) Eca-109 and (B) TE-1 cells were seeded into 6-well plates to adhere overnight, and then treated with 2 µg/ml DHA for 24 h. Levels of autophagy- and EMT-associated proteins were detected using western blot analysis and the relative density was analyzed using ImageJ software. Furthermore, 1×10⁶ (C) Eca-109 and (D) TE-1 cells were seeded into 6-well plates overnight and then transfected with CA-Akt. The cell monolayers were scratched with 1-ml tips after 12 h, subsequently cultured with medium containing 3% FBS, and then treated with 2 µg/ml DHA. Images of wound closure distances were captured after DHA treatment at 0 and 24 h. Scale bar, 100 µm. Following 12 h of transfection with CA-Akt, (E) Eca-109 and (F) TE-1 cells were treated with 2 µg/ml DHA for 24 h, and the levels of autophagy- and EMT-associated proteins were determined using western blot analysis and the relative densities were analyzed using ImageJ software. Data are presented as the mean ± standard deviation of 3 independent experiments. *P<0.05 and **P<0.01. DHA, dihydroartemisinin; p, phosphorylated, LC3, microtubule-associated protein 1A/1B-light chain 3; SQSTM, sequestosome 1; CA, constitutively active; EMT, epithelial-mesenchymal transition; -, without; +, with.