The effect of urotensin II on doxorubicin-induced apoptosis and its underlying mechanism in cardiomyocytes and human umbilical vein endothelial cells

• Main Authors: CHEN YEN-LING, 陳燕玲
• Other Authors: Tsai Chien-Sung
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/53526899840854612482

博士 === 國防醫學院 === 醫學科學研究所 === 103 === BACKGROUND AND PURPOSE Regulation of the homeostasis of vascular endothelium is critical for the processes of vascular remodeling and angiogenesis under physiological and pathological conditions. Urotensin II (U-II), a potent vasoactive peptide, participates in vascular and myocardial remodeling after injury. Recent studies indicate that U-II participates in vascular and myocardial remodeling after injury. The objective of this study was set to evaluate the effect of U-II on doxorubicin-induced human umbilical vein endothelial cells (HUVECs) and cardiomyocyte apoptosis, and to explore its intracellular mechanism(s). We investigated the protective effect of U-II on doxorubicin (DOX)-induced apoptosis in cardiomyocyte, cultured human umbilical vein endothelial cells (HUVECs) and the potential mechanisms involved in this process. EXPERIMENTAL APPROACH Rat neonatal cardiomyocytes and Cultured HUVECs were treated with vehicle, DOX (1 µM), U-II, or U-II plus DOX. HUVECs viability was determined by using 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyl tetrazolium (MTT, Sigma-Aldrich). Total RNA was extracted from HUVECs using the TRIzol method according to the protocol recommended by the manufacturer (Invitrogen, Carlsbad, CA, USA). Activity of caspase-3 was determined using a commercial kit (Promega; Madison, WI, USA) according to the manufacturer’s instructions. ATF3 siRNAs and mock control oligonucleotides were transfected using the Lipofectamine (Invitrogen) reagent according to the manufacturer’s instructions. Apoptosis was evaluated by DNA strand break level with TdT-mediated dUTP nick-end labeling (TUNEL) staining. Western blot analysis was employed to determine the related protein expression and flow cytometry assay was used to determine the TUNEL positive cells. KEY RESULTS Akt and ERK phosphorylation was enhanced by U-II treatment in cardiomyocytes. U-II-mediated Akt and ERK phosphorylation could be prevented by treatment with the U-II antagonist, urantide. Furthermore, U-II administration increased the cell viability and lowered apoptotic cell death induced by doxorubicin, as indicated by a decrease in trypan blue-positive cells, and reduced TUNEL staining and caspase-3 activity. These protective effects of U-II were abolished by prior inhibition of phosphatidylinositol 3-kinase/Akt by LY294002 (2-[4-morpholinyl]-8-phenyl-4H-1-benzopyran-4-one), and ERK by U0126 (1,4-diamino-2,3- dicyano-1,4-bis[2-aminophenylthio] butadiene). These findings suggest that U-II protects cardiomyocytes from doxorubicin-induced apoptosis partly via Akt and ERK, which may promote cell survival and cardioprotection. U-II prevents cardiomyocytes apoptosis via PI3K/ Akt and ERK. On the other hand, U-II reduced the quantity of cleaved caspase-3 and cytosol cytochrome c and increased Bcl-2 expression, which results in protecting HUVECs from DOX -induced apoptosis. U-II induced Activating transcription factor 3 (ATF3) at both mRNA and protein levels in U-II-treated cells. Knockdown of ATF3 with ATF3 siRNA significantly reduced ATF3 protein levels and U-II protective effect. U-II downregulated p53 expression in DOX-induced HUVECs apoptosis, and it rapidly activated extracellular signal-regulated protein kinase (ERK) and Akt. The inhibitory effect of U-II on DOX-increased apoptosis was attenuated by inhibitors of ERK (U0126) and PI3K/Akt (LY294002). CONCLUSION AND IMPLICATIONS Our observations provide evidence that U-II protects cardiomyocytes and HUVECs from DOX-induced apoptosis. U-II protects cardiomyocytes from DOX-induced apoptosis partly via Akt and ERK, which may promote cell survival and cardioprotection. In addition, U-II protects HUVECs from DOX-induced apoptosis, ERK-Akt phosphorylation, ATF3 activation, and p53 downregulation may play a signal-transduction role in this process.