Resveratrol analogue (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl) ethenyl]-quinazoline induces apoptosis via Fas-mediated pathway in HL-60 human leukemia cells

Abstract

Previously, we reported that (E)-8-acetoxy-2-[2-(3,4-diacetoxyphenyl)ethenyl]-quinazoline (8-ADEQ), a synthetic analogue of resveratrol had anti-inflammatory and G2/M cell cycle arrest activities, but the underlying molecular mechanism of cytotoxic effects of this compound was not determined. In this study, 8-ADEQ displayed potent cytotoxicity and triggered apoptosis in HL-60 cells as evidenced by DNA fragmentation, DNA ladder formation, and the externalization of Annexin V-targeted phosphatidylserine residues in HL-60 cells. In addition, 8-ADEQ triggered activation of caspases-8, -9, -6 and -3 and cleavage of their substrates such as poly(ADP-ribose) polymerase (PARP). Moreover, 8-ADEQ induced loss of mitochondrial membrane potential (MMP) and release of cytochrome c to the cytosol. Caspase-3 inhibitor (z-DEVD-fmk), caspase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD), and broad caspase inhibitor (z-VAD‑fmk) significantly suppressed the 8-ADEQ-induced DNA fragmentation. Interestingly, pretreatment with z-IETD-fmk, a caspase-8 inhibitor, completely abolished 8-ADEQ-induced caspase-3 and -9 activation, and subsequent DNA fragmentation. 8-ADEQ also increased the expression of Fas, Fas-associated death domain (FADD) and FasL, and formation of death-inducing signaling complex (DISC). Further analysis revealed that 8-ADEQ-induced apoptosis was mediated by upregulation of reactive oxidative species (ROS) generation. Taken together, our data indicated that 8-ADEQ-stimulated apoptosis in HL-60 leukemia cells is due to a Fas-mediated caspase-8-dependent pathway via ROS generation, but also, to a lesser extent cytochrome c release and caspase-9 activation.

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