Doxorubicin-induced apoptosis in H9c2 cardiomyocytes by NF-κB dependent PUMA upregulation.
OBJECTIVE
The cardiotoxicity of doxorubicin limits its clinical use in the treatment of a variety of solid tumors and malignant hematologic disease. Although the mechanism by which it causes cardiac injury is not yet known, apoptosis has been regarded as one of mechanisms underlying the cardiotoxic effects of doxorubicin. In the present study, we investigates the mechanisms of doxorubicin-induced apoptosis in H9c2 cardiomyocytes by NF-κB dependent PUMA upregulation.


METHODS
H9c2 cardiac myocytes was treated with 5-50 mM concentration of doxorubicin for 72 hours. p65 siRNA or PUMA siRNA was transfected into H9c2 cardiac myocytes, then treated with 50 mM doxorubicin for 72 hours. Apoptotic cells were detected using Flow cytometric analysis. MTT cytotoxicity test was used to detect the effect of doxorubicin on H9c2 cardiac myocytes. PUMA expression was detected by western blot assay. NF-kappaB binding activities was detected using electrophoretic mobility shift assays (EMSA).


RESULTS
Doxorubicin promotes apoptosis and inhibits proliferation in H9c2 cardiac myocytes in a dose and time-dependent way. Doxorubicin treatment increased NF-kappaB binding activities and PUMA expression. Inhibition of p65 by p65 siRNA decreased PUMA expression of doxorubicin-induced. Furthermore, p65 siRNA or PUMA siRNA-mediated suppression of p65 or PUMA results in inhibition of apoptosis of doxorubicin-induced.


CONCLUSIONS
It is demonstrated that PUMA is a direct target of NF-κB and mediates doxorubicin-induced apoptosis in vitro.
Department of Internal Medicine-Cardiovascular, The Affiliated Hospital of Medical College Qingdao University, Qingdao, Shandong, R.P. China. Anyi621@126.com.
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D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000235:genetics*; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage / Q000633:toxicity*; D024202:Electrophoretic Mobility Shift Assay; D005434:Flow Cytometry; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000473:pathology; D016328:NF-kappa B / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage; D051381:Rats; D013997:Time Factors; D051996:Transcription Factor RelA; D015854:Up-Regulation / Q000187:drug effects
D000903:Antibiotics, Antineoplastic; D051017:Apoptosis Regulatory Proteins; C533952:Bbc3 protein, rat; D016328:NF-kappa B; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D004317:Doxorubicin