Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors Giuseppina Raspaglio, Cristiano Ferlini, Simona Mozzetti, Silvia Prislei, Daniela Gallo, Nandita Das, Giovanni Scambia * Laboratory of Antineoplastic Pharmacology, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy Received 27 July 2004; accepted 10 September 2004 Abstract During a cellular screening of thiocolchicine analogs, thiocolchicine dimers resulted particularly active in cisplatin-resistant A2780- CIS cells. In order to discover by which mechanism(s) thiocolchicine dimers overcame cisplatin resistance, p53, p21 waf1 and MLH1 were assessed by Western blot. Results pointed out that, when combined with cisplatin, dimers increased the amount of all the three proteins with respect to the levels obtained by single drug exposure, thereby suggesting an interference in the process of repair of the cisplatin- induced DNA lesions. Moreover, in isolated nuclei drugs were able to produce DNA breaks, as demonstrated by Comet assay, thereby proving that the compounds were able to target cell nucleus independently from microtubules. Since Topo-I (topoisomerase I) is directly involvedin the DNA repair and such activity is overexpressed in cisplatin-resistant cells, Topo-I was investigated as a potential target. Using DNA relaxation assay, thiocolchicine dimers inhibited Topo-I, a property not shared by thiocolchicine. At variance with camptothecin, dimers did not produce cleavable complexes, thereby indicating that Topo-I inhibition occurs upstream of the religation step. To assess the mechanism of inhibition, an electrophoretic mobility shift assay between DNA and Topo-I was performed and revealed that thiocolchicine dimers specifically interfere with binding of Topo-I to DNA. The interference is specific since the same compounds did not modulate DNase activity and did not act as intercalating agents in the DNA unwinding assay. Finally, behaviour of dimers as spindle poisons was investigated and no relevant changes with respect to thiocolchicine in terms of interaction with microtubules were found. # 2004 Elsevier Inc. All rights reserved. Keywords: Thiocolchicine; Topoisomerase-I; Drug-resistance; Spindle poisons; DNA repair; Cisplatin 1. Introduction Colchicine is a plant alkaloid derived from autumn crocus (Colchicum autumnale) and is still one of the most effective treatments to relieve the intense pain associated with an acute gout attack. Such a potent anti-inflammatory activity is probably dependent on the fact that colchicine is able to inhibit microtubule dynamics, by hampering micro- tubule polymerisation [1]. The consequent microtubule paralysis blocks the release of pro-inflammatory mediators in leukocytes and other inflammatory cells [2]. In cancer cells microtubule-interacting drugs act as spindle poisons, thereby blocking the cell cycle at the M phase and inducing apoptosis [3]. This latter feature under- lies the antitumour properties belonging to such agents. Therefore, colchicine and its active analogues exhibit excellent antitumour properties ‘‘in vitro’’ and have been extensively investigated as potential anticancer drugs. However, neither colchicine nor its analogues have been extensively used as anticancer drugs, due to the extreme toxicities and the consequent unfavourable therapeutic index noticed in preclinical experimental models and in clinical trials. Colcemid (N-deacetyl-N-methylcolchicine) was the unique colchicine analogue used as anticancer drug, but was early replaced by Vinca alkaloids, which exhibited a superior therapeutic index in clinical studies. Colchicine framework (Fig. 1) includes a trimethoxy- phenyl (A ring), a seven membered ring (B ring) with an acetamide at the seven positions, and a tropolonic group (C ring). Thiocolchine (Fig. 1) differs from colchicine owing to the presence of a methylthio group instead of a methoxy at C10 in the tropolonic ring. In earlier studies, www.elsevier.com/locate/biochempharm Biochemical Pharmacology 69 (2005) 113–121 Abbreviations: EMSA, electrophretic mobility shift assay; Topo-I, topoisomerase-I; SSB, single strand break; NER, nucleotide excision repair; CPT, camptothecin * Corresponding author. Tel.: +39 06 35508736; fax: +39 06 35508736/3051160. E-mail address: cferlini@rm.unicatt.it (G. Scambia). 0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bcp.2004.09.004