Doxorubicin induced ROS-dependent HIF1α activation mediates blockage of IGF1R survival signaling by IGFBP3 promotes cardiac apoptosis

Abstract

Doxorubicin (Dox) causes the generation of intracellular reactive oxygen species (ROS) and inactivates insulin-like growth factor 1 (IGF1) signaling, leading to cardiomyocyte apoptosis. IGF-binding protein 3 (IGFBP3) is the most abundant circulating IGF1 carrier protein with high affinity, which has been reported to mediate ROS-induced apoptosis. Hypoxia-inducible factor 1α (HIF1A), an upstream protein of IGFBP3 is regulated by prolyl hydroxylase domain (PHD) through hydroxylation. In this study, we investigated the role of IGFBP3, HIF1A, and PHD in Dox-induced cardiac apoptosis.Cells challenged with 1 μM Dox for 24 h increased ROS generation, augmented intracellular and secreted IGFBP3 levels, and reduced IGF1 signaling. Further, we showed that Dox enhanced the extracellular association of IGF1 with IGFBP3. Moreover, echocardiography parameters, especially ejection fraction (EF) and fractional shortening (FS) were significantly reduced in ventricle tissue of Dox challenged rats. Notably, siRNA approach against IGFBP3 or an anti-IGFBP3 antibody rescued Dox-induced cardiac apoptosis, mitochondrial ROS, and the decrease in the IGF1 signaling activity. Furthermore, silencing HIF1A either using siRNA or inhibitor downregulated intracellular IGFBP3, rescued apoptosis, mitochondrial generation, and reduction in IGF1 signaling. Finally, western blot data revealed that ROS scavenger reversed Dox-induced cardiac apoptosis, increased levels of HIF1A and secreted IGFBP3, and decreased IGF1 survival signaling and PHD expression.These findings suggest that Dox-induced ROS generation suppressed PHD, which might stabilize nuclear HIF1A protein, leading to increased IGFBP3 expression and secretion. This in turn results in enhanced extracellular association of the latter with IGF1 and blocks IGF1 pro-survival signaling and may result in inducing cardiac apoptosis.

Keywords: IGF-binding protein-3; cardiomyocyte apoptosis; doxorubicin; hypoxia-inducible factor 1α; reactive oxygen species.

Figure 1

Doxorubicin increases generation of ROS and augments apoptosis in cardiac cells. H9c2 cells challenged with increasing doses of doxorubicin (Dox) for 24 h were harvested. (A) Apoptosis as detected using the TUNEL assay. (B) Mitochondrial superoxide production measured using MitoSOX staining. (C) The protein expression levels of the NADPH oxidase subunits NOX-2, p47, and p22 were detected using immunoblotting. (D) Protein levels of Bcl-xL, Bax, and cleaved caspase 3 were analyzed using western blot. Data are presented as mean ± standard deviation (n = 3). Scale bar represents 100 μm. Statistical significance is indicated as follows: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 2

Doxorubicin increases expression and secretion of IGFBP3, and decreases cardiac cell survival in vitro and in vivo. (A) H9c2 cells were incubated with Dox for 24 h at the indicated doses and protein expression of IGFBP3 and that of the components of survival signaling pathway were measured using immunoblotting. (B) Left ventricles of control and Dox-administered rats were isolated and the levels of IGFBP3 and survival-related proteins were analyzed using western blotting. (C) IGFBP3 expression from the rat left ventricle tissue was detected using immunohistochemistry. (D) H9c2 cells were challenged with Dox for 24 h at indicated doses and the amount of secreted IGFBP3 in both cell medium and in the animal serum of Dox-treated rats was examined using western blotting. The quantitative plot of IGFBP3 from the sera of doxorubicin challenged rats is included. Scale bar indicates 200 μm.

Figure 3

Doxorubicin enhances extracellular binding of IGFBP3 to IGF1, blocking IGFI survival signaling and promoting cell apoptosis. H9c2 cells were treated with 1.0 μM Doxorubicin (Dox) for 24 h. (A) Extracellular association of IGF1 with IGFBP3 was detected using co-IP. (B, C) Dox-challenged cells were incubated with either (B) anti-IGFBP3 antibody at indicated amounts or transfected with (C) Igfbp3 siRNA. The levels of pro-survival and apoptosis-related proteins were analyzed using western blotting. (D, E) H9c2 cardiomyoblasts challenged with Dox (1.0 μM) for 24 h were either incubated with anti-IGFBP3 antibody and/or transfected with Si-Igfbp3 and thereafter probed with TUNEL and MitoSOX reagents to evaluate the apoptotic cell death (D) and mitochondrial superoxide generation (E). Data are expressed as mean ± standard deviation (n = 3). Scale bar represents 100 μm. Statistical significance is showed as follows: ^*P < 0.05, ^**P < 0.01.

Figure 4

Dox-induced ROS production, cell apoptosis, downregulation of pro-survival signaling, and upregulation of IGFBP3 expression are dependent upon HIF1A activity. (A) H9c2 cells were incubated with increasing dose of Dox for 24 h. HIF1A protein levels were analyzed in H9c2 cells and animal cardiac tissue using western blotting. (B) Nuclear translocation of HIF1A in cells challenged with or without Dox were detected using fluorescence microscopy. Blue represents nucleus; green represents HIF1A. (C, D) Dox-exposed cells were treated with HIF1A inhibitor (C) or transfected with Hif1a siRNA (D). The levels of IGFBP3, pro-survival, and pro-apoptotic proteins in cells and cell culture medium (for secreted IGFBP3) were detected using western blotting. (E, F) Dox challenged H9c2 cells either treated with HIF1A inhibitor or transfected with siHif1a, were incubated with TUNEL and MitoSOX reagents to assess apoptosis mediated cell death (E) and mitochondrial ROS production (F). Statistical significance difference was shown as ^*P < 0.05, ^**P < 0.01 from at least three independent experiments.

Figure 5

Dox-induced mitochondrial ROS stabilizes HIF1A through the downregulation of PHD and promotes IGFBP3-induced cardiac apoptosis. (A) H9c2 cells and tissues from rats administered with indicated concentrations of Dox were subjected to western blotting. (B, C) Dox-exposed cells were treated with the ROS scavenger NAC (B), mitochondria complex I inhibitor rotenone (Rot) or the NADPH oxidase inhibitor Apo (C). The harvested cellular extract was analyzed using western blotting. (D) Cells were transfected with a PHD overexpression plasmid of the indicated amount and levels of HIF1α, IGFBP3, pro-survival, and pro-apoptotic proteins in cells and cell culture medium (for secreted IGFBP3) were measured by western blot analysis. Data represents as the mean ± standard deviation of the mean (n = 3). Statistical significance is represented as follows: ^*P < 0.05, ^**P < 0.01.

Figure 6

Graphical representation illustrating the mechanism underlying Dox-induced cell apoptosis. Dox induced ROS generation, which suppressed PHD. This stabilized nuclear HIF1A, promoted IGFBP3, and enhanced its extracellular association with IGF1. This interaction blocked survival signaling and resulted in cell apoptosis.