Abou-El-Hassan M A I, Rabelink M J W E, van der Vijgh W J F, Bast A, Hoeben R C
Department of Medical Oncology, Free University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
Br J Cancer. 2003 Dec 1;89(11):2140-6. doi: 10.1038/sj.bjc.6601430.
Cardiotoxicity is the main dose-limiting side effect of doxorubicin in the clinic. Being a free radical producer, doxorubicin affects the heart specifically because of its low antioxidant capacity. Among those antioxidants, catalase is present in very low levels in the heart compared to other organs. Since catalase is an essential enzyme in detoxifying hydrogen peroxide, the aim of the present study was to investigate the protective effect of catalase as delivered by an adenovirus vector against doxorubicin-induced cardiotoxicity in cultured neonatal rat cardiac myocytes (NeRCaMs). 7-Monohydroxyethylrutoside (MonoHER), a potent cardioprotector currently under clinical investigations, was included in the study as a reference. Neonatal rat cardiac myocytes were infected with different multiplicity of infections (MOIs) of adenovirus encoding catalase (AdCat). A control infection with an adenovirus vector encoding a nonrelated protein was included. The activity and content of catalase in infected cells were determined during 3 days postinfection. One group of NeRCaMs was infected with AdCat before treatment with doxorubicin (0-50 microM). The second and third group were treated with doxorubicin (0-50 microM) with and without 1 mM monohydroxyethylrutoside (monoHER), respectively. The LDH release and viability of treated cells were measured 24 and 48 h after doxorubicin treatment. The beating rate was followed in three other groups of cells receiving the same treatments within 3 days after doxorubicin (0-100 microM) treatment. Catalase activity increased in AdCat-infected cells, with different MOIs, starting from the second day after infection as compared to the mock-infected cells (P<0.03). At the third day of infection, an MOI of more than 50 caused cytopathic effects, which hampered the use of higher viral titres. With an MOI of 50, catalase activity increased 3.5-fold in AdCat-infected cells 3 days postinfection (P=0.021) compared to mock-infected cells. The beating rate and survival of NeRCaMs decreased in a concentration and time-dependent manner after doxorubicin treatment (P<0.0005). This cytotoxicity was associated with an increase in the LDH release from the treated cells (P<0.0005). The cells stopped beating 24 h after treatment with >50 microM doxorubicin. A 3.5-fold increase in the activity of catalase did not protect NeRCaMs against any of the cytotoxic effects of doxorubicin on NeRCaMs. In contrast, monoHER (1 mM) significantly protected NeRCaMs against the lethal effects of doxorubicin on the survival, LDH release and the beating rate of NeRCaMs (P<0.004) during 48 h after doxorubicin treatment. This protection resulted in a prolongation of the beating of doxorubicin-treated cells after the end of the experiment (i.e. >72 h). The present study (1) illustrates that the cytotoxicity of high MOI of AdCat (>50) limited the possibility to increase catalase activity more than 3.5-fold, which was not enough to protect infected NeRCaMs against doxorubicin-induced cardiotoxicity and (2) confirms the efficacy of monoHER as a cardioprotector. Thus, the use of monoHER proves more suitable for the prevention of doxorubicin-induced cardiotoxicity than catalase gene transfer employing adenovirus vectors.
心脏毒性是临床上阿霉素的主要剂量限制性副作用。作为一种自由基产生剂，阿霉素因其低抗氧化能力而对心脏有特异性影响。在那些抗氧化剂中，与其他器官相比，过氧化氢酶在心脏中的含量非常低。由于过氧化氢酶是解毒过氧化氢的一种必需酶，本研究的目的是研究腺病毒载体递送的过氧化氢酶对培养的新生大鼠心肌细胞（NeRCaMs）中阿霉素诱导的心脏毒性的保护作用。7-单羟乙基芦丁（MonoHER），一种目前正在临床研究中的有效心脏保护剂，被纳入研究作为对照。用不同感染复数（MOIs）的编码过氧化氢酶的腺病毒（AdCat）感染新生大鼠心肌细胞。包括用编码无关蛋白的腺病毒载体进行对照感染。在感染后3天内测定感染细胞中过氧化氢酶的活性和含量。一组NeRCaMs在用阿霉素（0 - 50 microM）处理前用AdCat感染。第二组和第三组分别在有和没有1 mM单羟乙基芦丁（monoHER）的情况下用阿霉素（0 - 50 microM）处理。在阿霉素处理后24小时和48小时测量处理后细胞的乳酸脱氢酶（LDH）释放和活力。在阿霉素（0 - 100 microM）处理后3天内，对接受相同处理的另外三组细胞监测其搏动率。与 mock 感染的细胞相比，AdCat 感染的细胞中过氧化氢酶活性从感染后第二天开始，在不同 MOIs 下均增加（P<0.03）。在感染第三天，MOI 大于50会引起细胞病变效应，这阻碍了使用更高的病毒滴度。MOI 为50时，感染 AdCat 的细胞在感染后3天过氧化氢酶活性比 mock 感染的细胞增加3.5倍（P = 0.021）。阿霉素处理后，NeRCaMs 的搏动率和存活率以浓度和时间依赖性方式下降（P<0.0005）。这种细胞毒性与处理后细胞中 LDH 释放增加有关（P<0.0005）。用>50 microM 阿霉素处理24小时后细胞停止搏动。过氧化氢酶活性增加3.5倍并不能保护 NeRCaMs 免受阿霉素对 NeRCaMs 的任何细胞毒性作用。相比之下，在阿霉素处理后48小时内，monoHER（1 mM）显著保护 NeRCaMs 免受阿霉素对其存活、LDH 释放和搏动率的致死作用（P<0.004）。这种保护导致在实验结束后（即>72小时）阿霉素处理的细胞搏动时间延长。本研究（1）表明高 MOI 的 AdCat（>50）的细胞毒性限制了将过氧化氢酶活性提高超过3.5倍的可能性，这不足以保护感染的 NeRCaMs 免受阿霉素诱导的心脏毒性，（2）证实了 monoHER 作为心脏保护剂的有效性。因此，与使用腺病毒载体进行过氧化氢酶基因转移相比，使用 monoHER 被证明更适合预防阿霉素诱导的心脏毒性。
