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Multiple actions of pifithrin-alpha on doxorubicin-induced apoptosis in rat myoblastic H9c2 cells
Chu Chang Chua1, Xuwan Liu, Jinping Gao
1Cardiovascular Research Laboratory, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA. chuac@etsu.edu
Abstract:
Doxorubicin (Dox) is a chemotherapeutic agent that causes significant cardiotoxicity. We showed previously that Dox activates p53 and induces apoptosis in mouse hearts. This study was designed to elucidate the molecular events that lead to p53 stabilization, to examine the pathways involved in Dox-induced apoptosis, and to evaluate the effectiveness of pifithrin-alpha (PFT-alpha), a p53 inhibitor, in blocking apoptosis of rat H9c2 myoblasts. H9c2 cells that were exposed to 5 muM Dox had elevated levels of p53 and phosphorylated p53 at Ser15. Dox also triggered a transient activation of p38, p42/p44ERK, and p46/p54JNK MAP kinases. Caspase activity assays and Western blot analysis showed that H9c2 cells treated with Dox for 16 h had marked increase in the levels of caspases-2, -3, -8, -9, -12, Fas, and cleaved poly(ADP ribose) polymerase (PARP). There was a concomitant increase in p53 binding activity, cytochrome c release, and apoptosis. These results suggest that Dox can trigger intrinsic, extrinsic, and endoplasmic reticulum-associated apoptotic pathways. Pretreatment of cells with PFT-alpha followed by Dox administration attenuated Dox-induced increases in p53 levels and p53 binding activity and partially blocked the activation of p46/p54JNK and p42/p44ERK. PFT-alpha also led to decreased levels of caspases-2, -3, -8, -9, -12, Fas, PARP, cytochrome c release, and apoptosis. Our results suggest that p53 stabilization is a focal point of Dox-induced apoptosis and that PFT-alpha interferes with multiple steps of Dox-induced apoptosis.
Insights
Doxorubicin (Dox) causes heart damage by activating p53 and apoptosis. Pifithrin-alpha (PFT-alpha) inhibits p53, reducing Dox-induced cell death and cardiotoxicity.
Area of Science:
- Cardiology
- Molecular Biology
- Pharmacology
Background:
- Doxorubicin (Dox) is a chemotherapy drug known for causing significant cardiotoxicity.
- Previous studies indicate Dox activates p53 and induces apoptosis in cardiac cells.
Purpose of the Study:
- To investigate the molecular mechanisms behind Dox-induced p53 stabilization and apoptosis.
- To evaluate the efficacy of pifithrin-alpha (PFT-alpha), a p53 inhibitor, in preventing Dox-induced apoptosis in rat H9c2 myoblasts.
Main Methods:
- H9c2 myoblasts were treated with Doxorubicin (Dox) and/or pifithrin-alpha (PFT-alpha).
- Assays included Western blot for protein levels (p53, caspases, PARP, Fas), MAP kinase activation, p53 binding activity, and apoptosis detection.
- Caspase activity was measured directly.
Main Results:
- Dox treatment increased p53 levels, phosphorylation, and binding activity, alongside activating MAP kinases (p38, ERK, JNK).
- Dox induced apoptosis via intrinsic, extrinsic, and ER-associated pathways, evidenced by increased caspases (-2, -3, -8, -9, -12), Fas, cleaved PARP, and cytochrome c release.
- PFT-alpha pretreatment attenuated Dox-induced p53 stabilization, partially blocked MAP kinase activation, and significantly reduced apoptosis markers.
Conclusions:
- p53 stabilization is a critical event in Doxorubicin-induced cardiotoxicity.
- Pifithrin-alpha effectively mitigates Dox-induced apoptosis by interfering with multiple apoptotic pathways, suggesting therapeutic potential.
