Arenobufagin activates p53 to trigger esophageal squamous cell carcinoma cell apoptosis in vitro and in vivo

Figures & data

Figure 1 In vitro anticancer activities of Bu and ArBu.

Notes: (A) Chemical structure of Bu and ArBu. (B) Cytotoxicity of Bu and ArBu toward human cancer and normal cells. The ESCC cells and Het-1A human esophageal squamous cells were treated with Bu and ArBu for 72 hours, and the cell viability was examined by MTT assay.
Abbreviations: Bu, bufalin; ArBu, arenobufagin; ESCC, esophageal squamous cell carcinoma.

Figure 2 Induction of Eca-109 cell apoptosis by ArBu.

Notes: Representative images of DNA fragmentation and nuclear condensation as examined by TUNEL–DAPI co-staining assay (magnification, 200×). The cells were treated with ArBu at 2 μM and 4 μM for 24 hours. Values in the images indicate the percentage of cell apoptosis induced by ArBu. Difference with P<0.01 (**) was considered statistically significant.
Abbreviations: ArBu, arenobufagin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3 Caspase activities of Eca-109 cells exposed to ArBu as examined using specific fluorogenic substrate.

Notes: *P<0.05; **P<0.01 versus the control. The cells were treated with ArBu at 2 μM and 4 μM for 24 hours.
Abbreviation: ArBu, arenobufagin.

Figure 4 Activation of p53 by ArBu.

Notes: (A) Phosphorylation of p53 at the sites of Ser15 and expression of total p53 in Eca-109 cells induced by ArBu for 24 hours. (B) Immunofluorescence of p-p53 in cells treated with ArBu for 24 hours (magnification, 600×).
Abbreviations: ArBu, arenobufagin; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 5 Downregulation of p53 by siRNA transfection (A) and the effects on cell apoptosis induced by ArBu (4 μM, 24 hours) as examined by PI flow cytometric analysis (B).

Notes: All results were obtained from three independent experiments. Bars with different characters (a, b, and c) are statistically different at P<0.05 level.
Abbreviations: siRNA, small interfering RNA; ArBu, arenobufagin; PI, propidium iodide.

Figure 6 In vivo antitumor efficacy and action mechanisms of ArBu.

Notes: (A, B) Inhibition of Eca-109 xenograft tumor weight and volume by ArBu (2 mg/kg or 4 mg/kg) for 20 days. *P<0.01 versus the control. (C) HE staining and immunohistochemical analysis of the expression levels of Ki-67, p-p53, and TUNEL in Eca-109 xenograft tumor treated with ArBu for 20 days.
Abbreviations: ArBu, arenobufagin; HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.