Identifying a Resistance Determinant for the Antimitotic Natural Products Disorazole C1 and A1 □S

Disorazoles are macrocyclic polyketides first isolated from the fermentation broth of the myxobacterium Sorangium cellulosum. Both the major fermentation product disorazole A1 and its much rarer companion disorazole C1 exhibit potent cytotoxic activity against many human tumor cells. Furthermore, the disorazoles appear to bind tubulin uniquely among known antimitotic agents, promoting apoptosis or premature senescence. It is uncertain what conveys tumor cell sensitivity to these complex natural products. Therefore, we generated and characterized human tumor cells resistant to disorazole C1. Resistant cells proved exceedingly difficult to generate and required single step mutagenesis with chronic stepwise exposure to increasing concentrations of disorazole C1. Compared with wild-type HeLa cells, disorazole C1-resistant HeLa/DZR cells were 34and 8-fold resistant to disorazole C1 and disorazole A1 growth inhibition, respectively. HeLa/DZR cells were also remarkably cross-resistant to vinblastine (280-fold), paclitaxel (2400-fold), and doxorubicin (47-fold) but not cisplatin, suggesting a multidrug-resistant phenotype. Supporting this hypothesis, MCF7/MDR cells were 10-fold cross-resistant to disorazole C1. HeLa/DZR disorazole resistance was not durable in the absence of chronic compound exposure. Verapamil reversed HeLa/DZR resistance to disorazole C1 and disorazole A1. Moreover, HeLa/DZR cells expressed elevated levels of the drug resistance ATP-binding cassette ABCB1 transporter. Loss of ABCB1 by incubation with short interfering RNA restored sensitivity to the disorazoles. Thus, the multidrug resistance transporter ABCB1 can affect the cytotoxicity of both disorazole C1 and A1. Disorazole C1, however, retained activity against cells resistant against the clinically used microtubulestabilizing agent epothilone B. The myxobacterium Sorangium cellulosum has been a rich source of pharmacologically interesting secondary metabolites, including the clinically used semisynthetic analog of epothilone B ixabepilone (Chin et al., 2006). Disorazole polyene macrodiolides were isolated first in 1994 and observed to have significant antifungal activity with no antibacterial activity (Jansen et al., 1994). Initial biochemical and pharmacological studies focused on the major fermentation product disorazole A1, which blocks cell proliferation, causes G2/M phase arrest and loss of microtubules, and induces apoptosis (Elnakady et al., 2004; Kopp et al., 2005). More recently, we synthesized and explored the actions of the minor fermentation component, disorazole C1, in part because it lacked the reactive epoxide found on disorazole A1 (Supplemental Fig. 1). Remarkably, disorazole C1 has potent antiproliferative activity against a wide variety of human tumor cells, it disrupts cellular microtubule integrity, it blocks microtubule polymerization in vitro, it binds tubulin in a unique manner, and it causes apoptosis and premature cellular senescence, all attributes associated with a promising anticancer agent (Wipf and Graham, 2004; Wipf et al., 2006; Tierno et al.,