To verify this hypothesis, cardiomyocytes were treated with 1 M doxorubicin, which had caused the most intense perturbation of IGF-1R and IGFBP-3 expression

A agent western blot for IGF-1R and IGFBP-three is shown in (D). , P <0.05 vs. Ctr , P <0.01 vs. Ctr , P <0.001 vs. Ctr. a, P <0.01 vs. Dox 0.5 b, P <0.05 vs. Dox 0.1 c, P <0.001 vs. Dox 0.1.As only IGF-1 that is not bound to IGFBP engages IGF-1R and exert biological effects [19], we determined the amount of free hormone two hours after adding 100 ng/mL IGF-1 to the culture media of doxorubicin-treated or control cardiomyocytes. As expected [18,19], the measured values were very small, being the result of IGF-1 degradation, sequestration by IGFBP-3 (and possibly other IGFBP), and interaction with IGF-1R that had occurred over the two hours. Consistent with the observed up-regulation of IGFBP-3 (Fig 1C), free IGF-1 levels progressively decreased in the media of cells that had been pre-incubated with 0.1 or 0.5 M doxorubicin compared to untreated ones (Fig 4A). Following exposure to 1 M doxorubicin, the concentration of free IGF-1 was also lower than in control media, but higher than the one reached with 0.5 M doxorubicin. This U-shape trend may be explained by the fact that 1 M doxorubicin not only induced IGFBP-3, but also significantly down-regulated IGF-1R (Fig 1B), thereby leading to the redistribution of free IGF-1 from the cell membrane to the culture medium. Akt is a main mediator of IGF-1 pro-survival intracellular signaling [7]. In agreement with the diminished availability and impaired anti-apoptotic action of IGF-1, pre-exposure to doxorubicin profoundly inhibited the phosphorylation of Akt elicited by IGF-1 (Fig 4B and 4C).Fig 2. Effect of exogenous IGF-1 on doxorubicin-induced apoptosis of H9c2 cells: annexin V/propidium iodide. Frequency of apoptotic cells, as assessed by annexin V/propidium iodide staining, 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with doxorubicin (Dox) IGF-1 at the indicated concentrations. , P <0.05 vs. Ctr , P <0.01 vs. Ctr , P <0.001 vs. Ctr. e, P <0.01 vs. Dox 0.1 P <0.05 vs. IGF-1 0.01.Serine phosphorylation of IRS-1, an adaptor protein that lies downstream of IGF-1R, may interrupt IGF-1 signaling cascade [7], but this was not the case with doxorubicin in H9c2 cells (Fig 4B and 4C).Since wild-type p53 is a transcriptional repressor of IGF1R [20] and an inducer of IGFBP3 [21], we reasoned that the modifications in IGF-1R and IGFBP-3 levels triggered by doxorubicin might be mediated by p53. Indeed, doxorubicin dose-dependently caused the accumulation of p53 in H9c2 cells (Fig 5A), as already demonstrated [22]. To verify this hypothesis, cardiomyocytes were treated with 1 M doxorubicin, which had caused the most intense perturbation of IGF-1R and IGFBP-3 expression, after being incubated with the p53 inhibitor, PFT-. Pretreatment with PFT prevented the decrease in IGF-1R and the increase in IGFBP-3 induced by8198578 doxorubicin (Fig 5B). In addition, it 136553-81-6 reduced apoptosis (Fig 5C).