Human TDP1 ELISA Matched Antibody Pair

Tirapazamine (TPZ) is an anticancer drug with highly selective cytotoxicity toward hypoxic cells. TPZ is converted to a radical intermediate under hypoxic conditions, and this intermediate interacts with intracellular macromolecules, including DNA. TPZ has been reported to indirectly induce DNA double-strand breaks (DSBs) through the formation of various intermediate DNA lesions under hypoxic conditions. Although the topoisomerase II DNA complex has been identified as one of these intermediates, other lesions have not yet been defined. In order to obtain a deeper understanding of the mechanisms responsible for the selective cytotoxicity of TPZ toward hypoxic cells, its cellular sensitivity was systematically examined with genetically isogenic DNA-repair-deficient mutant DT40 cell lines. Our results showed that tdp1(-1-), td2(-/-) parp1(-/-), and aptx1(-/-) cells displayed hypersensitivity to TPZ only under hypoxic conditions. These results strongly suggest that the accumulation of the topoisomerase I-trapped DNA complex, topoisomerase II-trapped DNA complex, and abortive ligation products with 5'-AMP are the potential causes of TPZ-induced hypoxic cell death. Furthermore, our genetic analysis revealed that under normoxic conditions (as well as hypoxic conditions), TPZ exhibited significant cytotoxicity toward cell lines deficient in homologous recombination, nonhomologous end joining, base excision repair, and translesion synthesis. Ascorbic acid, a radical scavenger, suppressed TPZ-induced cytotoxicity toward normoxic cells. These results suggest the involvement of oxidative DNA damage and DSBs produced by reactive oxygen species generated from superoxide, a byproduct of the oxidation of TPZ radical intermediates in normoxic cells. Collectively, our results demonstrate that TPZ induces oxidative DNA damage under normoxic and hypoxic conditions and selectively introduces abortive topoisomerase DNA complexes and unligatable DNA ends under hypoxic conditions.

A polysaccharide (termed ACPS-1) from mycelia of Antrodia cinnamomea under submerged culture was purified by hot water extraction and successive DEAE-52 cellulose and Sephadex G-100 column chromatography, and structurally characterized by FTIR, NMR, periodate oxidation, Smith degradation, and GC-MS. ACPS-1 (MW 2.296 x 10(4) Da) was composed primarily of Man, Xyl, Ara, Fuc and Rha with a molar ratio of 31.27:1.77:1.44:1.34:1.00, and its backbone consisted of repeating alpha-(1 -> 3), alpha-(1 -> 6), alpha-(1 -> 2), and alpha-(1 -> 4) glycosidic linkages. ACPS-1 displayed strong in vitro growth-inhibitory effects on several human and mouse cancer cell lines (HeLa, A431, H22 and 5180), and were not cytotoxic to normal mouse spleen cells. Studies of the inhibitory mechanism revealed that ACPS-1 induced apoptosis and cell cycle arrest (cells remained in G2/M phase) through blocking of topoisomerase I/tyrosyl-DNA phosphodiesterase I (TOP1/TDP1)-mediated DNA repair pathway. Our findings suggest that ACPS-1 has strong potential applications in pharmaceutical and food industries, and as a novel anticancer agent based on its dual TOP1/TDP1 inhibitory effect. (C) 2018 Elsevier B.V. All rights reserved.