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Doxorubicin (DOX), an antineoplastic agent widely used for the treatment of cancer, belongs to the anthracycline family of antitumor antibiotics. DOX may undergo one-electron reduction to the corresponding semiquinone free radical by flavin-containing reductases. Under aerobic conditions, the semiquinone radical reacts rapidly with oxygen to generate superoxide anion, undergoing redox cycling. At moderate concentrations, reactive oxygen species (ROS) play an important role as regulatory mediators in signaling processes. We have shown that DOX increased phosphorylation of enzymes comprising mitogen-activated protein (MAP) kinase cascades in primary hepatocyte cultures, and that this action was independent of oxidant damage. In particular, extracellular signal-regulated kinase (ERK) was phosphorylated by the drug treatment. In this work, we have determined the possible involvement of particular free radicals in DOX-induced ERK phosphorylation in hepatocyte cultures by using specific free radical scavengers. The levels of ERK phosphorylation were measured by Western blot analysis with an anti-Thr202/Tyr204-phosphorylated p44/p42 MAPK antibody. Deferoxamine (DFO; iron chelator), catalase (hydrogen peroxide-removing enzyme), or alpha-tocopherol (peroxyl-radical scavenger) did not affect DOX-increased ERK phosphorylation levels. However, the cell-permeable superoxide dismutase mimetic MnTBAP and the flavin-containing enzyme inhibitor diphenyleneiodonium reverted DOX-induced effects. These results suggest that superoxide anions, probably generated by DOX metabolism, are involved in the effects of the anthracycline on the MAP kinase cascade activation.

Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited on account of its toxicity. DOX cytotoxic effects have been associated with reactive oxygen species (ROS) generated during drug metabolism. ROS induce signaling cascades leading to changes in the phosphorylation status of target proteins, which are keys for cell survival or apoptosis. The mitogen-activated protein kinase (MAPK) cascades are routes activated in response to oxidative stress. In this work, the effects of DOX on cytotoxicity, indicators of oxidative stress (malondialdehyde -MDA- and GSH), and the phosphorylation status of extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and p38 kinases were analyzed in primary cultures of rat hepatocytes. DOX (1–50 μM) did not modify lactate dehydrogenase (LDH ) release into the medium, the levels of MDA (determined by high-performance liquid chromatography [HPLC]) or the intracellular GSH during the incubation time up to 6 h. GSH levels from mitochondria extracted by Percoll gradient from cultured hepatocytes were not modified by DOX, thus excluding its depletion or any impaired mitochondrial uptake. Characterization of proteins by Western blot analysis revealed that DOX increased phosphorylation of p38 kinases and JNK1 and JNK2 in a dose- and time-dependent manner. DOX also increased ERK2 phosphorylation at latter time points. In conclusion, DOX triggers activation of ERK, JNK, and p38 kinases in primary cultures of rat hepatocytes independently of oxidant damage. [ABSTRACT FROM AUTHOR]

Abstract: Doxorubicin (DOX) is a potent anticancer drug, whose clinical use is limited due to its toxicity. This toxicity has been associated with free radicals generated during the drug metabolism. We previously found that DOX increased the intracellular diacylglycerol (DAG) levels at 1h in isolated rat hepatocytes, probably by mobilizing choline-enriched phospholipids. In this work, we studied the effects of DOX on oxidative stress markers, and the possible contribution of ceramide metabolism to DAG accumulation. Other possible routes of DAG production, such as impairment of triacylglycerol (TAG) synthesis, and their connection with oxidative stress were also investigated. Time-course experiments revealed that DOX decreased intracellular GSH at 2h, but did not affect cell viability, ATP or malondialdehyde (MDA) levels at any time. DOX did not modify the intracellular levels of [3H]-ceramide during the first 90min of exposure, but increased it significantly at 2h. [3H]-Sphingomyelin remained unchanged during the whole period. These results indicate that ceramide metabolism is not involved in the early DAG response to DOX. The drug markedly increased the incorporation of [3H]-oleate into intracellular DAG from 60min. In contrast, DOX reduced the incorporation of [3H]-oleate into intracellular phospholipids and TAG. DOX inhibited TAG synthesis at the DAG acyltransferase step. These results suggest that DOX increases the intracellular levels of the lipid messengers, ceramide and DAG, by independent mechanisms. Activation of the de novo synthesis of ceramide is probably involved in the sphingolipid accumulation, while inhibition of TAG synthesis contributes to DAG accumulation, this response being independent of oxidative damage. [Copyright &y& Elsevier]