A Cysteine-Reactive Alkyl Hydroquinone Modifies Topoisomerase IIα, Enhances DNA Breakage, and Induces Apoptosis in Cancer Cells

We previously reported that the anticancer activity of a botanical compound 10'(Z),13'(E),15'(E)-heptadecatrienylhydroquinone [HQ17(3)] was attributed to topoisomerase (Topo) II alpha poisoning and the induction of oxidative damage. HQ17(3) irreversibly inhibits Topo II alpha activity in vitro and is more cytotoxic in leukemia HL-60 cells than in Topo II alpha-deficient variant HL-60/MX2 cells, which suggests that Topo II alpha is a cellular target of HQ17(3). This study further characterizes the molecular mechanisms of the anticancer activity of HQ17(3). Proteomic analyses indicated that HQ17(3) reacted with Cys-427, Cys-733, and Cys-997 of recombinant Topo II alpha in vitro, whereas it reacted with Cys-427 of cellular Topo II alpha in Huh7 hepatoma cells. The modification of HQ17(3) inhibited Topo II alpha catalytic activity, increased the Topo II alpha-DNA cleavage complex, and caused the accumulation of DNA breakage. In Huh7 cells, HQ17(3) treatment caused prompt inhibition of DNA synthesis and consequently induced the expression of DNA damage-related genes DDIT3, GADD45A, and GADD45G. Topo II alpha inhibition, apoptosis, and oxidative stress were found to account for cytotoxicity caused by HQ17(3). Pretreatment of Huh7 cells with N-acetylcysteine (NAC) partially attenuated mitochondrial membrane damage, DNA breakage, and caspase activation. However, NAC pretreatment did not diminish HQ17(3)-induced cell death. These results suggest that the anticancer activity of HQ17(3) is attributed significantly to Topo II alpha poisoning. The structural feature of HQ17(3) can be used as a model for the design of Topo II alpha inhibitors and anticancer drugs.