阿霉素通过 NF-κB 依赖性 PUMA 上调诱导 H9c2 心肌细胞凋亡。

Doxorubicin-induced apoptosis in H9c2 cardiomyocytes by NF-κB dependent PUMA upregulation.

机构信息

Department of Internal Medicine-Cardiovascular, The Affiliated Hospital of Medical College Qingdao University, Qingdao, Shandong, R.P. China.

出版信息

Eur Rev Med Pharmacol Sci. 2013 Sep;17(17):2323-9.

PMID:24065225

Abstract

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.

MATERIALS AND 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.

摘要

目的

阿霉素的心脏毒性限制了其在治疗多种实体瘤和恶性血液病中的临床应用。虽然其导致心脏损伤的机制尚不清楚，但凋亡已被认为是阿霉素心脏毒性作用的机制之一。本研究通过 NF-κB 依赖性 PUMA 上调来研究阿霉素诱导 H9c2 心肌细胞凋亡的机制。

材料和方法

用 5-50mM 浓度的阿霉素处理 H9c2 心肌细胞 72 小时。将 p65 siRNA 或 PUMA siRNA 转染到 H9c2 心肌细胞中，然后用 50mM 阿霉素处理 72 小时。用流式细胞术分析检测凋亡细胞。MTT 细胞毒性试验用于检测阿霉素对 H9c2 心肌细胞的影响。用 Western blot 检测 PUMA 表达。用电泳迁移率变动分析（EMSA）检测 NF-κB 结合活性。

结果

阿霉素以剂量和时间依赖的方式促进 H9c2 心肌细胞凋亡和抑制增殖。阿霉素处理增加 NF-κB 结合活性和 PUMA 表达。用 p65 siRNA 抑制 p65 可降低阿霉素诱导的 PUMA 表达。此外，p65 siRNA 或 PUMA siRNA 介导的 p65 或 PUMA 抑制导致阿霉素诱导的凋亡抑制。