Abstract & Overview
OBjECTIvE:To examine the modulatory effects of the antioxidant Trolox in the interplay of Akt and apoptosis signal-regulating kinase (Ask) 1 in doxorubicin (Dox)-induced oxidative stress (OS).METHODS: Rat cardiomyocytes were treated with Dox (5 µM) or Trolox (20 µM) for 24 h, or were pretreated with Trolox (20 µM) for 4 h before treatment with Dox (5 µM) for 24 h, and were compared with cardiomyocytes without any treatment.For the Akt inhibition study, cardiomyocytes were pretreated overnight with the phosphoinositol 3-kinase/Akt inhibitor wortmannin (1 µM) before treatment with Dox, Trolox or Trolox + Dox.The gain and loss of Akt gene function with HA-tagged constitutively active Akt1 or dominant-negative mutant Akt1 (T308A; S473A) was also studied in the cardiomyocytes under all treatment conditions.Cells exposed to various treatment conditions were analyzed for OS, apoptotic and antiapoptotic proteins, and phosphorylation of Ask1 Ser83 , Ask1 Thr845 and Akt Ser473 .RESuLTS: Dox caused a decrease in the phosphorylation of Akt Ser473 .The phosphorylation of Ask1 was differentially modulated by Dox: Ask1 phosphorylation was upregulated at Thr845 sites and was downregulated at Ser83 sites.The levels of apoptotic proteins were also increased.Trolox pretreatment downregulated Dox-induced OS, promoted the activation of Akt Ser473 , and modulated Dox-induced changes in the phosphorylation of Ask1 and apoptosis-related proteins.Inhibition of phosphoinositol 3-kinase/Akt using wortmannin and dominant-negative Akt1 adenovirus promoted phosphorylation of Ask Thr845 and attenuated the phosphorylation of Ask1 Ser83 .In contrast, upregulation of Akt activity using constitutively active Akt1 promoted phosphorylation of Ask1 Ser83 and inhibited phosphorylation of Ask1 Thr845 , respectively.COnCLuSIOnS: Suppression of the effects of Dox by Trolox in rat cardiomyocytes occurs through the upregulation of Akt and its consensus site Ask1 Ser83 .