Subcellular distribution of epothilones in human tumor cells

Abstract

Epothilones are a new class of natural and potent antineoplastic agents that stabilize microtubules. Although 12,13-epoxide derivatives are potent antiproliferative agents, the activities of the corresponding 12,13-olefin analogs are significantly decreased. These data were confirmed for two new analogs, 6-propyl-EpoB (pEB) and 6-propyl-EpoD (pED), in comparison with the natural compounds EpoB͞EpoD, by using human A431, MCF7, and MDR1-overexpressing NCI͞Adr cells. By using tritiated pEB͞pED, compound uptake, release, and nuclear accumulation were investigated in A431 and NCI͞Adr cells. In these cells, epothilones can principally be recognized and exported by Verapamil-sensitive efflux pumps, which are not identical to MDR1. The degree of export depends on the structure, olefin vs. epoxide-analog, and also on the intracellular drug concentration. The accumulation of pED used at 3.5 or 70 nM, respectively, was increased in the presence of 10 M Verapamil in both cell lines 2-to 8-fold. In contrast, the intracellular levels of pEB were affected by Verapamil only at 3.5 nM pEB in NCI͞Adr (2-fold) and not in A431 cells. In addition, strong nuclear accumulation was observed for pEB (40 -50%) but not paclitaxel or pED (5-15%) in both cell lines. Our study suggests that differences in growth inhibitory efficacy between epoxide and olefin analogs may be based on different mechanisms of drug accumulation and subcellular distribution. Epothilones are a new class of natural products and potential antineoplastic agents. Although they have no structural similarities to taxanes, they exert cellular effects similarly to paclitaxel (Taxol). Thus, epothilones bind to tubulin and cause hyperstabilization of microtubules with subsequent mitotic arrest and apoptotic cell death (1-3). The molecular mechanisms by which epothilones and paclitaxel induce apoptosis remain to be elucidated.It has been demonstrated that epothilones are generally superior to paclitaxel in their ability to inhibit the proliferation of human cancer cell lines that are resistant to commonly used anticancer agents, including paclitaxel. The best understood mechanism of resistance to cytotoxic drugs, including antimicrotubule agents is drug export by multidrug-resistant pglycoprotein (MDR1) (4). Although taxanes are substrates for MDR1, epothilones are not, and thus MDR1-positive tumor cells remain sensitive to epothilones (1, 2, 5-9).The 12,13-epoxide moiety of epothilone A (EpoA) and B (EpoB) is dispensable for tubulin͞microtubule-related effects in vitro, because the corresponding olefin analogs (EpoC and EpoD), as inducers of tubulin polymerization, are equally potent to EpoA and EpoB (7,8,10). Formal removal of epoxide oxygen in EpoB, thus leading to EpoD, does cause a significant decrease (ϳ10-to 30-fold) in antiproliferative activity (9). Furthermore, exposure of tumor cells to EpoB for a 4-h period produces virtually the same growth inhibitory effect as a continuous 3-day exposure, whereas for EpoD, exposure times more than 4 h were required to produce ef...

Radiosensitizing effect of epothilone B on human epithelial cancer cells

“…First, γH2AX foci assays showed that ionizing radiation combined with epothilone B resulted in a concentration-dependent increase of the number of double-strand breaks which suggests a reduction in DNA repair capacity. Lichtner et al [27] published their observation of a strong accumulation of epothilone B in the cell nucleus, more precisely in the fraction of nuclear proteins. This indicates an inhibition of proteins like the nuclear tubulin isotype [27] which, thus, results in a possible DNA-damaging effect of epothilone B [9].…”

“…Lichtner et al [27] published their observation of a strong accumulation of epothilone B in the cell nucleus, more precisely in the fraction of nuclear proteins. This indicates an inhibition of proteins like the nuclear tubulin isotype [27] which, thus, results in a possible DNA-damaging effect of epothilone B [9]. Swanton et al [41] emphasized that the effect of microtubulestabilizing agents is based on the repression of genes which play a role in DNA repair (e.g., BRCA1, H2AFX).…”

Improved Cellular Pharmacokinetics and Pharmacodynamics Underlie the Wide Anticancer Activity of Sagopilone

“…The pan caspase inhibitor benzyloxycarbonylVal-Ala-Asp(OMe).fluoromethylketone (Z-VAD-fmk) was purchased from Bachem AG. Stock solutions were prepared and stored as previously detailed (18).…”

“…This suggests that some epothilones may overcome multidrug resistance (MDR) mechanisms, which would augment their therapeutic potential. Natural epothilones, however, are not optimal candidates for anticancer therapy for a number of reasons, including a limited therapeutic index and poor tolerability (18)(19)(20). Because of this, novel epothilone derivatives have been synthesized and investigated in preclinical models (17)(18)(19)(20).…”

Patupilone (epothilone B) inhibits growth and survival of multiple myeloma cells in vitro and in vivo

“…[25][26][27] In contrast to paclitaxel, epothilones in general are equally cytotoxic against paclitaxelsensitive and paclitaxel-resistant human cancer cell lines, including those overexpressing MDR1/Pgp. 11,[13][14][15][16]28,29 In addition, although point mutations in the major expressed ␤-tubulin isoform result in a 24-fold increase in resistance to paclitaxel, these same mutations result in only a 1.3-to 4-fold cross-resistance to patupilone. 28 To date, patupilone has been evaluated in 2 phase 1 studies, and several phase 2 clinical trials are ongoing.…”