All-trans retinoic acid suppresses the angiopoietin-Tie2 pathway and inhibits angiogenesis and metastasis in esophageal squamous cell carcinoma

Fig 1. ATRA treatment shows a time-dependent and dose-dependent inhibition on cell viability. EC1 cells were cultured in RPMI-1640 supplemented with 10% FBS and seeded in 96 well plates. (A) After 0 hours, 24 hours and 48 hours, the number of live cells was counted in ATRA and fluorouracil treatment groups. (B) The proportion of dead cells were calculated and presented as Percent of death cells/total number of cells. (C) After 24 hours, ATRA and fluorouracil show additive effect on cell proliferation. (D) After 24 hours, ATRA caused apoptosis of cells. Densitometry analysis of protein levels of Cl-PARP. (E) The effect of ATRA and fluorouracil on the expression of Cl-Caspase3. (F) Densitometry analysis of protein levels of Cl-caspase3. *p<0.05; **p<0.01; *** p<0.001. Student t-test.

Fig 2. ATRA treatment reduces cell migration in EC1 cells. EC1 cells were cultured in RPMI-1640 supplemented with 10% FBS and seeded in 6 well plates. Scratches on cell monolayer were made using pipette tips when cells became confluent. Cells were then treated with 3 concentrations of ATRA (0.1, 1, 10 μmol/L), fluorouracil (100 mg/L), or untreated for 24 hours. Images were chosen from 10 random fields to calculate the average distances. Data were presented as average length of cell-free void ± SD. (B) Representative pictures of wound healing assay. *p<0.05; **p<0.01; *** p<0.001. Student t-test.

Fig 3. The transcript levels of Angiopioteins-Tie-2 pathway are downregulated in EC1 cells. EC1 cells were treated with ATRA at 0.1, 1, or 10 μmol/L, 100 mg/L fluorouracil, 10 μmol/L AM80, 100 mg/L fluorouracil plus 10 μmol/L AM80, or untreated for 24 hours. RNA was isolated from treated cells. Real-time RT-PCR analysis was performed to assessed the transcript levels of (A) Ang-1, Ang-2 and Tie-2. (B) Ang-1. (C) Ang-2. (D) Tie-2. (E) VEGF. (F) Flt-1. (G) KDR. *p<0.05; **p<0.01; *** p<0.001. Student t-test.

Fig 4. ATRA treatment decreases the expression of Ang-1, Ang-2 and Tie-2 in EC1 cells. EC1 cells were treated with 3 concentrations of ATRA (0.1, 1, 10 μmol/L), fluorouracil (100 mg/L) for 24 hours, or untreated. (A) The protein levels of Ang-1, Ang-2 and Tie-2 were examined using western blot. Densitometry analysis of the protein levels of Ang-1, Ang-2 or Tie-2 (B); Ang-1 (C); Ang-2(D); and Tie-2 (E). β-actin was used as a loading control. *p<0.05; **p<0.01; *** p<0.001. Student t-test.

Fig 5. ATRA treatment suppresses the growth of xenograft tumors of EC1 cells and improves the cachexia of mice. (A) 1x106 EC1 cells were subcutaneously injected into mice at both flanks on day 0. Ten days post-cell inoculation, mice bearing xenograft tumors were randomized to five groups and treated for 10 days with placebo, fluorouracil (50 mg/kg/day), or 3 concentrations of ATRA (0.1, 1, or 10 mg/kg/day). Mice were killed on day 20. Mouse body weight was measured before and after cells implantation, also before and after treatment. (B) The cachexia was recorded in mice treated with ATRA, fluorouracil, or placebo. Cachexia was assessed by body weight loss. (C) Images of tumors isolated from mice treated with ATRA and fluorouracil. (D) Average tumor size was calculated and shown in panel C. (E) Immunohistochemical staining of CD31, Ang-1, Ang-2 and Tie-2 in subcutaneous tumors. *p<0.05; **p<0.01; *** p<0.001. Student t-test.

Fig 6. Schematic illustration of ATRA effect on cell viability and angiogenesis in esophageal carcinoma. ATRA treatment induces cell apoptosis by controlling cell death regulators (survivin and bcl-2) via RAR-β, inhibits VEGF-VEGFR signaling pathway via RAR-α, and regulates the expression of angiogenic factors (Ang-1, Ang-2 and Tie-2) and angiogenesis. In vivo, ATRA suppressed tumor growth and metastasis. Previously reports and the current study demonstrate that ATRA inhibits cell viability, angiogenesis and metastasis of esophageal cancer cells in vitro and in vivo.