5-azacytidine, a chemotherapeutic drug, induces TRAIL-mediated apoptosis in mouse thymocytes in vivo

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL.