Abstract
Backgrounds. Doxorubicin (DOX) is an effective therapeutic drug for malignant tumors; however, its clinical applications were limited by its side effects, especially the cardiotoxicity caused by ROS-mediated p53 and MAPK signal pathways' activation-induced cell apoptosis. Sanyang Xuedai mixture (SYKT) has been reported as an antioxidant agent and attenuated DOX-induced cardiotoxicity by targeting ROS-mediated apoptosis, but the mechanisms are still not fully delineated. Objective. This study aimed at investigating whether SYKT alleviated DOX-induced cardiotoxicity by inhibiting ROS-mediated apoptosis and elucidating the role of ROS-mediated p53 and MAPK signal pathways' activation in this process. Materials and Methods. Identification, separation, and culture of mouse primary cardiomyocytes. Cells were treated with DOX (1 μM), SYKT (30 mg/mL), or SYKT coupled with DOX. The p53 inhibitor Pifithrin-α (PFT-α), p38/MAPK inhibitor SB203583 (SB), and JNK inhibitor SP600125 (SP) were used as positive control. Western blot was employed to detected p53 and p38 as well as JNK expressions and the activation and translocation of Bax and cytochrome C. Flow cytometer (FCM) was used to detect the mitochondrial membrane potential and cell apoptosis. Results. After separation and culture, 95% of cells showed positive cTnI expression, which indicated that mouse primary cardiomyocytes were successfully identified in our research. DOX activated p53 and MAPK signal pathways in a time-dependent manner, which were inactivated by being cotreated with SYKT, PFT-α, or SB, respectively. DOX significantly decreased Bax and increased cytochrome c expressions in the cytoplasm, whereas Bax was upregulated and cytochrome c was downregulated in the mitochondria, which were reversed by SYKT treatment. Besides, DOX reduced mitochondria membrane potential (MMP) in cardiomyocytes compared to the control group; SYKT recovered its MMP and attenuated DOX-induced cardiomyocyte injury. Of note, DOX increased the expression levels of cleaved caspase-3 as well as poly ADP-ribose polymerase (PARP) and promoted cell apoptosis, which were also reversed by SYKT treatment. Discussion and Conclusions. Our results indicated that SYKT alleviated DOX-induced cardiotoxicity by inhibiting p53 and MAPK signal pathways' activation-mediated apoptosis, and it might serve as a potential therapeutic agent for DOX-induced cardiotoxicity.
Highlights
Doxorubicin (DOX) has been widely used as a chemotherapeutic agent for various cancers’ treatment because of its high therapeutic efficacy and broad-spectrum effects; DOX-induced acute and chronic irreversible cardiotoxicities for patients limited its applications to a large extent [1]
Cell apoptosis was detected and the results showed that DOX increased reactive oxygen species (ROS)-mediated cardiomyocytes apoptosis in a time-dependent manner, which was reversed by coculturing with SYKT
Our further results showed that the expression levels of apoptosisassociated proteins including the cleaved caspase-3 and poly ADP-ribose polymerase (PARP) were elevated; interestingly, membrane potential (MMP) was reduced by DOX treatment; as a result, cytochrome c was released from mitochondria into cytoplasm, which verified that DOX induced ROS-mediated cell apoptosis, and these results were in accordance with the previous study [25]; by coculturing SYKT and DOX with cells, we found that SYKT reversed these phenomena
Summary
Doxorubicin (DOX) has been widely used as a chemotherapeutic agent for various cancers’ treatment because of its high therapeutic efficacy and broad-spectrum effects; DOX-induced acute and chronic irreversible cardiotoxicities for patients limited its applications to a large extent [1]. Recent studies have reported that DOX promoted reactive oxygen species (ROS) production in cells, which induced cell intoxication and played an important role in DOX induced cardiotoxicity [2, 3], but the mechanisms are still not fully delineated. High levels of ROS have been reported to induce apoptosis and necroptosis in human pancreatic cancer cells [4] and promoted cell death in HepG2 cells [5]. MAPK and p53 signal pathways played important roles in this process; high levels of ROS promoted cell autophagy. ROS might induce cell intoxication by activating MAPK and p53 signal pathways, which promoted cell autophagy and apoptosis. Our preliminary experiments found that DOX induced cell death and cardiotoxicity in a ROS-production-dependent manner [10], which was in accordance with previous studies. The studies above indicated that eliminating ROS might be an ideal method to alleviate DOX-induced cell intoxication
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