Hemiasterlins are sponge-derived tripeptides that inhibit cell growth by depolymerizing existing microtubules and inhibiting microtubule assembly. Since hemiasterlins are poor substrates for
P-glycoprotein, they are attractive candidates for
cancer therapy and have been undergoing clinical trials. The basis of resistance to a synthetic analogue of
hemiasterlin,
HTI-286 (HTI), was examined in cell populations derived from ovarian
carcinoma (A2780/1A9) cells selected in
HTI-286. 1A9-HTI-resistant cells (1A9-HTI(R) series) were 57-89-fold resistant to HTI. Cross-resistance (3-186-fold) was observed to other
tubulin depolymerizing drugs, with collateral sensitivity (2-14-fold) to
tubulin polymerizing agents. Evaluation of the percentage of polymerized and soluble
tubulin in 1A9 parental and 1A9-HTI(R) cells corroborated the HTI cytotoxicity data. At 22 degrees C or 37 degrees C, in the absence of any
drug, the percentage of polymerized microtubules for each of the 1A9-HTI(R) populations was greater than that in the 1A9 parental cells, consistent with more stable microtubules. Furthermore, microtubules in the 1A9-HTI(R) populations were also more resistant to depolymerization at 4 degrees C and had more acetylated and detyrosinated (Glu-
tubulin)
alpha-tubulin, all characteristic of more stable microtubules. The 1A9-HTI(R) cell populations exhibited either a single
nucleotide change in the
M40 beta-tubulin isotype, S172A, or in two cell populations where no
beta-tubulin mutation was detected, mutations in the Kalpha-1
alpha-tubulin isotype, S165P and R221H in one resistant cell population and I384V in another. Unlike reports of mutations resulting in reduced
drug affinity, the experimental data and location of mutations are consistent with resistance to
HTI-286 mediated by microtubule-stabilizing mutations in beta- or
alpha-tubulin.