BIBR1532 inhibits proliferation and metastasis of esophageal squamous cancer cells by inducing telomere dysregulation

Figure 1 IC₅₀ determination of BIBR1532 in esophageal squamous cell carcinoma cells. A and B: Viability of KYSE150 (A) and KYSE410 (B) cells after treatment with the indicated concentrations of BIBR1532 for 48 hours; C and D: Viability of KYSE150 (C) and KYSE410 (D) cells after treatment with the indicated concentrations of BIBR1532 for 72 hours.

Figure 2 Effect of BIBR1532 on the proliferation of KYSE150 and KYSE410 cells. A: Human telomerase reverse transcriptase expression in KYSE150 and KYSE410 cells after exposure to a series of BIBR1532 concentrations; B: Viability of KYSE150 and KYSE410 cells after treatment with various concentrations of BIBR1532 at the indicated time points; C: Plate colony formation by KYSE150 and KYSE410 cells after treatment with various concentrations of BIBR1532 at the indicated time points. ^aP < 0.05 was compared with the control group respectively. P < 0.05 was considered statistically significant. hTERT: Human telomerase reverse transcriptase.

Figure 3 Effect of BIBR1532 on the migration of KYSE150 and KYSE410 cells. A: Migration of KYSE150 and KYSE410 cells after treatment with various concentrations of BIBR1532 for 24 hours, as evaluated by scratch assay; B: Migration of KYSE150 and KYSE410 cells after treatment with various concentrations of BIBR1532 for 24 hours, as evaluated by the transwell assay.

Figure 4 Effect of BIBR1532 on the senescence of KYSE150 and KYSE410 cells. A: Senescence of KYSE150 and KYSE410 cells after treatment with various concentrations of BIBR1532 for 48 hours; B: Expression of the cell senescence biomarker p53 in KYSE150 and KYSE410 cells after treatment with various BIBR1532 concentrations for 48 hours.

Figure 5 Proliferation and migration of KYSE150 cells after treatment with BIBR1532 combined with human telomerase reverse transcriptase overexpression. A: Plate colony formation by KYSE150 cells after treatment with BIBR1532 (50 μM) and/or human telomerase reverse transcriptase (hTERT) overexpression for 48 hours; B: Migration evaluated by transwell assay of KYSE150 cells after treatment with BIBR1532 (50 μM) and/or hTERT overexpression for 48 hours; C: Migration evaluated by the scratch assay of KYSE150 cells after treatment with BIBR1532 (50 μM) and/or hTERT overexpression for 48 hours. hTERT: Human telomerase reverse transcriptase.

Figure 6 Effect of BIBR1532 on shelterin protein and key proteins in the DNA damage response pathway expression in KYSE150 and KYSE410 cells. A and B: Expression of telomeric-repeat binding factor 1 (TRF1), TRF2, TIN2-interacting protein 1, protection of telomeres 1, TIN2, and repressor activation protein 1 in KYSE150 (A) and KYSE410 (B) cells after treatment with indicated concentrations of BIBR1532 for 48 hours; C and D: Expression of γ-H2AX, p-ATM, CHK2, p-ATR, and CHK1 in KYSE150 (C) and KYSE410 (D) cells after treatment with the indicated concentrations of BIBR1532 for 48 hours. TRF1: Telomeric-repeat binding factor 1; TRF2: Telomeric-repeat binding factor 2; TPP1: TIN2-interacting protein 1; POT1: Protection of telomeres 1; TIN2: TRF1-interacting nuclear protein 2; RAP1: Repressor activation protein 1; γ-H2AX: Phosphorylated histone H2AX; p-ATM: Phosphorylated ataxia-telangiectasia mutated gene; p-ATR: Phosphorylated ataxia telangiectasia and Rad3-related protein; CHK2: Check point kinase 2; CHK1: Check point kinase 1.

Figure 7 Anti-cancer effect of BIBR1532 in an esophageal squamous cell carcinoma xenograft mouse model. A-C: Tumor images (A), growth curves (B), and tumor weights (C) obtained from xenograft tumors derived from KYSE150 cells treated with BIBR1532 (50 mg/kg); D and E: Expression of human telomerase reverse transcriptase, Ki-67, and γ-H2AX in xenografted tumor tissues evaluated by immunohistochemistry (D) and Western blotting (E); F: Macroscopic images of the heart, liver, spleen, lungs, and kidneys of nude mice treated with BIBR1532 or DMSO; G: Hematoxylin and eosin staining of the heart, liver, spleen, lung, and kidney tissues from nude mice treated with BIBR1532 or DMSO. ^aP < 0.05 was compared with the control group respectively. P < 0.05 was considered statistically significant. hTERT: Human telomerase reverse transcriptase; γ-H2AX: Phosphorylated histone H2AX.