Korean Circ J.  2008 May;38(5):264-269. 10.4070/kcj.2008.38.5.264.

Effects of Cardiotrophin-1 on Adriamycin-Induced Apoptosis in H9c2 Cardiomyoblasts

Affiliations
  • 1Department of Medicine, Sungkyunkwan University School of Medicine, Cardiac and Vascular Center, Samsung Medical Center, Seoul, Korea. esjeon@smc.samsung.co.kr

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 cyto-protective 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 micrometer of ADR), and ADR was shown to decrease procaspase-3. On RT-PCR, expression of Bax-alpha mRNA increased and Bcl-2 decreased during the 24 hours after ADR treatment. Consequently, the ratio of Bax-alpha/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-alpha 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.
CONCLUSION
These data demonstrated that ADR-induced apoptosis of cardiomyocytes can be prevented by CT-1; therefore, it may be possible to use CT-1 as a cardioprotective agent during ADR chemotherapy in patients with cancer.

Keyword

Adriamycin; Cardiotrophin-1; Apoptosis; Cell protection

MeSH Terms

Apoptosis
Caspase 3
Cell Count
Cell Death
Cell Survival
Cytoprotection
Doxorubicin
Heart
Humans
Myocytes, Cardiac
Phosphorylation
RNA, Messenger
Caspase 3
Doxorubicin
RNA, Messenger

Figure

  • 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.

  • Fig. 5 CT-1 phophorylated Akt. H9c2 cells were exposed to CT-1 (1 ng/mL) for the indicated times. Representative western blot for p-Akt are shown. CT-1: cardiotrophin-1.


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