Arenobufagin induces apoptosis in acute leukemia cells by activating Rho A/ROCK1 signaling pathway

Abstract

Objective To investigate the apoptosis-inducing effect of arenobufagin (ARE) on acute leukemia cells and explore its possible molecular mechanism. Methods Acute leukemia cell lines Jurkat and MV-4-11 were treated with different concentrations of ARE (0, 10, 20, 40 or 80 nmol/L) for 24 h, or with 80 nmol/L ARE for different durations (0, 6, 12, 18 or 24 h), or with 80 nmol/L ARE for 24 h after being pretreated with 20 μmol/L Y-27632 (inhibitor of ROCK1) for 1 h, respectively. Then, MTT assay and flow cytometry were used to respectively detect the effect of ARE on cell proliferation and apoptosis in acute leukemia cells; Western blotting and co-immunoprecipitation (CoIP) assay were performed to determine the expression of apoptosis-related and Rho A/ROCK1 pathway-related proteins in the acute leukemia cells after ARE treatment. Results ARE inhibited the proliferation and induced apoptosis of acute leukemia cells in dose- and time-dependent manners (P < 0.01). Western blotting results showed that ARE activated ROCK1 signaling pathway, facilitated the translocation of BAX from cytoplasm to mitochondria, promoted the release of mitochondrial proteins (Cytochrome c, AIF), increased the cleavage/activation of Caspase 3 and Caspase 7 as well as the degradation of Poly(ADP-ribose) polymerase 1 (PARP1), and then reduced the expression of X-linked inhibitor of apoptosis (XIAP). The above effects presented dose- and time-dependence (P < 0.01). Interruption of ROCK1 pathway by the pharmacological inhibitor Y-27632 significantly attenuated ARE-mediated mitochondrial translocation of BAX, reduced cytosolic release of Cytochrome c and AIF from mitochondria, suppressed the activation of Caspase 3 and Caspase 7, the degradation of cleavage of PARP1, and down-regulated XIAP expression as well as blocked apoptosis (P < 0.01). Conclusion ARE effectively induces apoptosis of acute leukemia cells (Jurkat, MV-4-11) through the activation of Rho A/ROCK1 signaling pathway.

Keywords

• arenobufagin
• acute leukemia
• rho-associated kinase 1
• apoptosis