Metallothionein Prevents Doxorubicin-induced Cardiotoxicity Via JAD-STAT Pathway In Cardiomyocytes

Dox (doxorubicin) is an anthracycline antibiotic and one of the most important anticancer agents. But it can easily accumulate in the heart tissue and then induce cardiac toxicity which can lead to severe myocardial injury and heart failure, thus this severely limits its clinical application. Most studies reported that the mechanism of Dox-induced cardiotoxicity is mainly attributed to the formation of reactive oxygen species (ROS). The excess of ROS attack myocardial cells, then cause myocardial oxidative damage. It has been reported that Dox can increase ROS generation in cardiac myocytes and decrease antioxidant enzyme activity in myocardial cell, so that the myocardial cells are in oxidative stress status.Metallothionein (MT) is a low-molecular weight, cysteine-rich, metal-binding protein which is widely present in various tissues and organs in mammals. MT can combine with heavy metals and function as antioxidant on scavenging free radicals. MT has a protective effect for tissue injury caused by exogenous substances in the state of oxidative stress. In previous studies, MT has been shown to be an effective protector against Dox-induced cardiomyopathy, but the exact mechanisms are still unknown.The Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway transmits information through transmembrane receptors, directly to target gene promoters in the nucleus, providing a mechanism for transcriptional regulation. JAK proteins are cytosolic tyrosine kinases and have four family members:JAK1, JAK2, JAK3and tyrosine kinase2(TYK2). JAK1and JAK2are expressed ubiquitously in many tissues whereas JAK3and TYK2are expressed in hematopoietic cells and in certain carcinoma cells. AG490, which is the Jak2inhibitor, is a PTK inhibitor and can be used as a specific inhibitor of JAK-STAT signaling pathway. There are seven STAT genes in mammalian cells, STAT1, STAT2, STAT3, STAT4, STAT5A,STAT5B and STAT6. JAK-STAT pathway played a critical role in the ischemia-reperfusion injury, cardiac hypertrophy, apoptosis, heart transplant and oxidative stress. In the previous experiment, we found that, on the gene expression of JAK-STAT pathway, there are some difference between MT-null mice (MT-/-) and wild-type mice (MT+/+) after Dox treatment. We thus hypothesize that MT may prevent Dox-induced cardiotoxicity involving JAK-STAT pathway. The objective of this study was to investigate the role of JAK-STAT pathway in MT protection of Dox-induced cardiotoxicity.In the present study, we established a primary culture system of cardiomyocytes from MT-/-and MT+/+mice and assayed JAK2and STAT3proteins and their phosphorylation of protein expression with Western blotting analysis. The results indicated that p-Jak2and p-Stat3levels in MT+/+cardiomyocytes were significantly higher than in MT’ cardiomyocytes when they were treated with Dox. These findings strongly support that JAK-STAT pathway can play the critical role during the protection of MT against Dox-induced cytotoxicity.In order to further study, we have joined the AG490which is the specific inhibitor of the JAK-STAT pathway. The experiment was divided into four groups:control group, AG490alone group, Dox alone group, and AG490+Dox group. To reflect myocardial cell damage, we observed the cardiomyocytes morphological alterations and measured the cardiac myocyte apoptosis by LDH leakage and Hoechst33258staining method. It was found that the use of AG490pretreatment can increase the number of cardiomyocyte apoptosis and myocardial cytotoxic in MT+/+cardiomyocytes.Numerous studies show that the cardiotoxicity of Dox is caused by the formation of ROS. The excess of ROS can attack lipids, proteins and DNA and other biological macromolecules, then result in oxidative damage of myocardial cells. In order to evaluate the role of MT prevents Dox-induced oxidative damage, this experiment reflect the antioxidant status of the myocardial cells through the myocardial intracellular levels of ROS, superoxide dismutase (SOD) vitality, catalase (CAT) vitality, and the level of lipid peroxidation and protein carbonylation. Our results showed that AG490can increase the Dox-induced oxidative damage and reduce the oxidative defense capability in MT+/+cardiomyocytes.To summarize, blocking the JAK-STAT pathway can increase the Dox-induced myocardial cell injury, reduce the antioxidant capacity of the myocardial cells and aggravate the Dox-induced oxidative injury in MT+/+cardiomyocytes, which were not dramatic in MT-/-cardiomyocytes. These findings strongly demonstrate that MT can prevent Doxorubicin-induced cardiotoxicity, which partially involves JAK-STAT signaling pathway.