A comparative study between catalase gene therapy and the cardioprotector monohydroxyethylrutoside (MonoHER) in protecting against doxorubicin-induced cardiotoxicity in vitro.
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.
Abou-El-Hassan
M A I
MA
Department of Medical Oncology, Free University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands. m.abdulhassan@vumc.nl
Rabelink
M J W E
MJ
van der Vijgh
W J F
WJ
Bast
A
A
Hoeben
R C
RC
D000256:Adenoviridae; D000818:Animals; D000831:Animals, Newborn; D000903:Antibiotics, Antineoplastic / Q000009:adverse effects*; D002316:Cardiotonic Agents / Q000627:therapeutic use*; D002374:Catalase / Q000235:genetics* / Q000378:metabolism; D002470:Cell Survival; D004317:Doxorubicin / Q000633:toxicity*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006331:Heart Diseases / Q000139:chemically induced / Q000517:prevention & control*; D006896:Hydroxyethylrutoside / Q000627:therapeutic use*; D007770:L-Lactate Dehydrogenase / Q000096:biosynthesis; D032383:Myocytes, Cardiac / Q000382:microbiology; D051381:Rats
D000903:Antibiotics, Antineoplastic; D002316:Cardiotonic Agents; D006896:Hydroxyethylrutoside; D004317:Doxorubicin; D007770:L-Lactate Dehydrogenase; D002374:Catalase