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Abstract
Background and Objectives
Adriamycin (doxorubicin, ADR) is a highly effective anti-neoplastic drug, but its clinical use is limited by its adverse side effects on the heart. Cardiotrophin (CT-1), a potent cardiac survival factor, is capable of inhibiting apoptosis in cardiac myocytes. The aim of this study was to investigate the cytoprotective effects of CT-1 against ADR-induced apoptosis in vitro.
Materials and Methods
We determined a reasonable ADR concentration for inducing cell death by utilizing a cell survival test performed in a dose-dependent manner. To determine the requirements for apoptosis in ADR-treated cardiac myocytes (H9c2 cells), we examined the effect of CT-1 on survival and apoptotic changes using a cell counting kit (CCK), RT-PCR, and Western blotting.
Results
In analyzing cell survival as determined by CCK, ADR-induced cell death was found to occur in a dose-dependent manner (50% death at 24 hours after 2 μM of ADR), and ADR was shown to decrease procaspase-3. On RT-PCR, expression of Bax-α mRNA increased and Bcl-2 decreased during the 24 hours after ADR treatment. Consequently, the ratio of Bax-α/Bcl-2 mRNA peaked at 24 hours after ADR treatment. In contrast, CT-1 effectively attenuated the ADR-induced cell death in a dose-dependent manner. The changes in Bax-α and Bcl-2 mRNA expression after ADR treatment were reversed by CT-1 (1 ng/mL) treatment. The protein levels of procaspase-3 decreased after ADR treatment, an effect which was reversed by CT-1 treatment. Akt phosphorylation was also increased by CT-1, demonstrating that CT-1 inhibited apoptosis induced by ADR.
Figures and Tables
Fig. 1
Cell viability test performed at 24 hr after treatment with ADR (A), with or without CT-1 (B). ADR induced cell death in a dose dependent manner (Panel A) and CT-1 prevent ADR-induced cell death from the concentration of 1 ng/mL. Result presented in bar graph are mean±SEM of three experiments. *p<0.01 when compared with control cells. ADR: adriamycin, CT-1: cardiotrophin-1.
Fig. 2
Bax-α and Bcl-2 mRNA expression in H9c2 after ADR 2 μM treatment. Normalization relative to GAPDH was performed. Mean mRNA levels in untreated control cells are expressed as 1. *p<0.01 when compared to control cells. CON: untreated control cells, ADR: Adriamycin, GAPDH: Glyceride 3-phosphate dohydroqenase.
Fig. 3
Bax-α and Bcl-2 mRNA expression in H9c2 after ADR (2 μM) and CT-1 (1 ng/mL) treatment. Normalization relative to GAPDH was performed. The mean mRNA levels in untreated control cells are expressed as 1. *p<0.01 when compared with control cells. CON: untreated control cells, ADR: Adriamycin, A+C: Adriamycin 2 μM and Cardiotrophin-1 1 ng/mL, CT-1: cardiotrophin-1, GAPDH: Glyceride 3-phosphate dohydroqenase.
Fig. 4
ADR induces apoptosis in H9c2. A: H9c2 cells were treated with 10 μM ADR for various time and analyzed by western blotting using anti-caspase-3 antibody. B: H9c2 cells were treated with ADR (10 μM) and CT-1 (500 ng/mL). ADR induces apoptosis were decreased by CT-1 treatment. α-tubulin level was also examined as a loading control. ADR: adriamycin, CT-1: cardiotrophin-1, CON: untreated control cells.
Acknowledgments
This work was supported by the Samsung Biomedical Research Institute grant, #SBRI C-A5-123-1.
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