Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors.
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, p21waf1 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 involved in 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.
Raspaglio
Giuseppina
G
Laboratory of Antineoplastic Pharmacology, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy.
Ferlini
Cristiano
C
Mozzetti
Simona
S
Prislei
Silvia
S
Gallo
Daniela
D
Das
Nandita
N
Scambia
Giovanni
G
D002460:Cell Line; D003078:Colchicine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D059004:Topoisomerase I Inhibitors
D004791:Enzyme Inhibitors; D059004:Topoisomerase I Inhibitors; C014856:thiocholchicine; D004264:DNA Topoisomerases, Type I; D003078:Colchicine