Peloruside- and Laulimalide-Resistant Human Ovarian Carcinoma Cells Have βI-Tubulin Mutations and Altered Expression of βII- and βIII-Tubulin Isotypes

Abstract

Peloruside A and laulimalide are potent microtubule-stabilizing natural products with a mechanism of action similar to that of paclitaxel. However, the binding site of peloruside A and laulimalide on tubulin remains poorly understood. Drug resistance in anticancer treatment is a serious problem. We developed peloruside A- and laulimalide-resistant cell lines by selecting 1A9 human ovarian carcinoma cells that were able to grow in the presence of one of these agents. The 1A9-laulimalide resistant cells (L4) were 39-fold resistant to the selecting agent and 39-fold cross-resistant to peloruside A, whereas the 1A9-peloruside A resistant cells (R1) were 6-fold resistant to the selecting agent while they remained sensitive to laulimalide. Neither cell line showed resistance to paclitaxel or other drugs that bind to the taxoid site on β-tubulin nor was there resistance to microtubule-destabilizing drugs. The resistant cells exhibited impaired peloruside A/laulimalide-induced tubulin polymerization and impaired mitotic arrest. Tubulin mutations were found in the βI-tubulin isotype, R306H or R306C for L4 and A296T for R1 cells. This is the first cell-based evidence to support a β-tubulin–binding site for peloruside A and laulimalide. To determine whether the different resistance phenotypes of the cells were attributable to any other tubulin alterations, the β-tubulin isotype composition of the cells was examined. Increased expression of βII- and βIII-tubulin was observed in L4 cells only. These results provide insight into how alterations in tubulin lead to unique resistance profiles for two drugs, peloruside A and laulimalide, that have a similar mode of action.

Low-dose laulimalide represents a novel molecular probe for investigating microtubule organization

“…1 The site is situated on the exterior of the MT on β-tubulin near the charged C-terminal tail, which was recently validated with the aid of macrolide-resistant cell lines. 2,3 Laulimalide exhibits microtubule-stabilizing activity and presents new opportunities for the development of antimitotic therapies. 4 It is highly cytotoxic, inhibiting cell proliferation in numerous cancer cell lines at low nM IC 50 values.…”

Development of a Novel Class of Tubulin Inhibitors with Promising Anticancer Activities

“…Although many MIAs are actively used in the clinic against a wide variety of solid tumors and hematological malignancies, many limitations of currently approved agents are encountered, including variable sensitivity of different cancers, innate and acquired drug resistance, side effects of peripheral neuropathy and neutropenia, and poor solubility that necessitates their clinical use (4,9). In addition to the development of overexpression of drug efflux pumps (24), mutations in the genes encoding a-and b-subunits of tubulin and differential expression of tubulin isotypes that reduce the binding of a drug to tubulin also contribute to MIA resistance (25,26). Therefore, discovery of new MIAs such as our HA14-1 analogs may be helpful in overcoming these problems and might offer advantages over the current agents used for chemotherapy.…”

βI-tubulin mutations in the laulimalide/peloruside binding site mediate drug sensitivity by altering drug–tubulin interactions and microtubule stability

“…Transfected cells were lysed and analysed by 10% SDS-PAGE and Western blotting at 72 h post-transfection as previously described [33]. Mouse monoclonal antibody against β-tubulin (3:1000, T4026, Sigma) was used.…”

“…By generating two PLA/LAU-resistant cell lines, we recently provided the first biological evidence that R306 and A296 of βI-tubulin were important for the binding of these MSAs to tubulin [33]. As with the taxoid site mutations [17,18], the R306H/C and A296T mutations acquired by the PLA and LAU-resistant cells led to an impaired ability of PLA and LAU to induce tubulin polymerization and block cells in mitosis, unless high concentrations of the compounds were added [33].…”

“…As with the taxoid site mutations [17,18], the R306H/C and A296T mutations acquired by the PLA and LAU-resistant cells led to an impaired ability of PLA and LAU to induce tubulin polymerization and block cells in mitosis, unless high concentrations of the compounds were added [33]. Supporting the notion that these drugs occupy a non-taxoid site location on β-tubulin, the mutant cells showed normal sensitivity to drugs that bind to taxoid-, vinca-or colchicine-sites on β-tubulin.…”