The taccalonolides are a class of structurally and mechanistically distinct microtubule-stabilizing agents isolated from Tacca chantrieri. A crucial feature of the taxane family of microtubule stabilizers is their susceptibility to cellular resistance mechanisms including overexpression of P-glycoprotein (Pgp), multidrug resistance protein 7 (MRP7), and the BIII isotype of tubulin. The ability of four taccalonolides, A, E, B, and N, to circumvent these multidrug resistance mechanisms was studied. Taccalonolides A, E, B, and N were effective in vitro against cell lines that overexpress Pgp and MRP7. In addition, taccalonolides A and E were highly active in vivo against a doxorubicin-and paclitaxel-resistant Pgp-expressing tumor, Mam17/ADR. An isogenic HeLa-derived cell line that expresses the BIII isotype of tubulin was generated to evaluate the effect of BIII-tubulin on drug sensitivity. When compared with parental HeLa cells, the BIII-tubulin-overexpressing cell line was less sensitive to paclitaxel, docetaxel, epothilone B, and vinblastine. In striking contrast, the BIII-tubulinoverexpressing cell line showed greater sensitivity to all four taccalonolides. These data cumulatively suggest that the taccalonolides have advantages over the taxanes in their ability to circumvent multiple drug resistance mechanisms.

Tubulin is the target for some very powerful anti-mitotic and anti-tumor drugs. The betaIII tubulin isotype is found in very few normal tissues, but is often found in tumors, where it has been implicated in resistance to anti-tumor drugs. The betaIII isotype occurs in fish, amphibians, birds and mammals and its unique features are highly conserved in evolution. One of these features is the replacement of cys239 by ser239. Cys239 is unusual in being highly sensitive to oxidation; in fact, oxidation of this residue inhibits microtubule assembly. The betaIII isotype also has a very unusual cys124, where other beta isotypes have ser/ala124. The striking conservation in betaIII of vertebrates strongly suggests that cys124 and ser239 play functional roles. We have prepared the C124S and S239C mutants of betaIII and tested their effects on the functional properties of tubulin. We have found that both the betaIII C124S and betaIII S239C mutants bind colchicine less well than does wild-type alphabetaIII, and also make transfected HeLa cells more resistant to colchicine. However, the double mutant, betaIII C124S/S239C, binds colchicine still less well than do either of the single mutants, but in contrast to the former, the double mutant increases the cells' sensitivity to colchicine. Our results indicate that the roles that these residues play in colchicine binding and microtubule integrity are far more complex than previously imagined and that the specific residues at which betaIII differs from the other isotypes act collectively to keep betaIII in a functional conformation.

The C termini of ␤-tubulin isotypes are regions of high sequence variability that bind to microtubule-associated proteins and motors and undergo various post-translational modifications such as polyglutamylation and polyglycylation. Crystallographic analyses have been unsuccessful in resolving tubulin C termini. Here, we used a stepwise approach to study the role of this region in microtubule assembly. We generated a series of truncation mutants of human ␤I and ␤III tubulin.

Supplementary Figure 1 from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms

Supplementary Figure 1 from The Taccalonolides: Microtubule Stabilizers That Circumvent Clinically Relevant Taxane Resistance Mechanisms