Fig 1.
Doxorubicin stimulates apoptosis and modulates IGF-1R/IGFBP-3 expression in H9c2 cells.
Frequency of apoptotic cells (A) and IGF-1R (B) and IGFBP-3 (C) expression (densitometry of western blot bands) 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). A representative western blot for IGF-1R and IGFBP-3 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.
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.
Fig 3.
Effect of exogenous IGF-1 on doxorubicin-induced apoptosis of H9c2 cells: TUNEL and caspase 3/7 activity.
Frequency of apoptotic cells, as assessed by TUNEL (A; representative microphotographs are shown in B) and fluorescence (AUF) produced by the cleavage of a substrate of activated caspase 3/7 (C), 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with doxorubicin (Dox) ± IGF-1 at the indicated concentrations. ***, P <0.001 vs. Ctr. c, P <0.001 vs. Dox 0.1; d, P <0.001 vs. Dox 0.5; e, P <0.01 vs. Dox 0.1; f, P <0.05 vs. Dox 0.5. ¥, P <0.001 vs. Dox 0.1 + IGF-1 100; ¢, P <0.001 vs. Dox 0.1 + IGF-1 100 and Dox 0.5 + IGF-1 100.
Fig 4.
Doxorubicin affects IGF-1 free levels and intracellular signaling.
(A) Concentrations of free IGF-1 measured in culture media two hours after adding 100 ng/ml IGF-1 to H9c2 cardiomyocytes that had not been treated (Ctr) or had been incubated with 0.1, 0.5, or 1 μM doxorubicin (Dox). (B and C) IGF-1 stimulated phosphorylation of Akt and IRS-1 (B, representative western blots; C, band densitometry) in H9c2 cardiomyocytes that had not been treated (Ctr) or had been incubated with 0.1, 0.5, or 1 μM Dox. ***, P <0.001 vs. Ctr. A, P <0.05 vs. IGF-1 100; B, P <0.001 vs. IGF-1 100; C, P <0.01 vs. IGF-1 100. ¶, P <0.01 vs. Dox 0.1 + IGF-1 100 and Dox 1 + IGF-1 100; §, P <0.001 vs. Dox 0.1 + IGF-1 100, Dox 0.5 + IGF-1 100, Dox 1 + IGF-1 100.
Fig 5.
Involvement of p53 in the change in IGF-1R /IGFBP-3 levels caused by doxorubicin.
(A) Representative western blot and band densitometry for p53 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 0.1, 0.5, or 1 μM doxorubicin (Dox). (B and C) IGF-1R/IGFBP-3 expression (band densitometry and representative western blot, B) and annexin V/propidium iodide positivity (C) in H9c2 cardiomyocytes untreated or exposed to 1 μM Dox with or without pre-treatment with PFT-α. *, P <0.05 vs. Ctr; ***, P <0.001 vs. Ctr. ^, P <0.01 vs. Dox 1.
Fig 6.
Antioxidants reverse doxorubicin-initiated apoptosis and IGF-1R /IGFBP-3 perturbation.
Frequency of apoptotic cells (A) and IGF-1R and IGFBP-3 expression (representative western blot in B and densitometry of western blot bands in C) 24 hours after no treatment (Ctr) or incubation of H9c2 cardiomyocytes with 1 μM doxorubicin (Dox) with or without pre-treatment with N-acetylcysteine (NAC), dexrazoxane (DEX), or carvedilol (CARV). *, P <0.05 vs. Ctr; **, P <0.01 vs. Ctr; ***, P <0.001 vs. Ctr; ****, P <0.0001 vs. Ctr. #, P <0.05 vs. Dox 1; ^, P <0.01 vs. Dox 1.