Novel suicide ligands of tubulin arrest cancer cells in S-phase

Abstract

It is presently accepted that the mechanism of action for all anti-tumor tubulin ligands involves the perturbation of microtubule dynamics during the G2/M phase of cell division and subsequent entry into apoptosis [1]. In this report, we challenge the established dogma by describing a unique mechanism of action caused by a novel series of tubulin ligands, halogenated derivatives of acetamido benzoyl ethyl ester. We have developed a suicide ligand for tubulin, which covalently attaches to the target and shows potent cancericidal activity in tissue culture assays and in animal tumor models. These compounds target early S-phase at the G1/S transition rather than the G2/M phase and mitotic arrest. Bcl-2 phosphorylation, a marker of mitotic microtubule inhibition by other tubulin ligands was dramatically altered, phosphorylation was rapid and biphasic rather than a slow linear event. The halogenated ethyl ester series of derivatives thus constitute a unique set of tubulin ligands which induce a novel mechanism of apoptosis.

Figure 1

Core structure of compounds. Structure of HAABE (A) and HAABU (B) (Halogen = F, Cl, Br, I).

Figure 2

Microtubule polymerization inhibition by IAABE and BAABE. Compounds were dissolved in G-PEM buffer at 4°C before resuspending the lyophilized tubulin in the wells of a 96-well plate (CytoDYNAMIX^™ Screen 01; Cytoskeleton Inc.). Absorbance was measured over time; absorbance is proportional to microtubule content. IC₅₀ values were determined by linear regression analysis of concentration versus percentage inhibition; concentration at 50% inhibition at 60 minutes was defined as the IC₅₀. Concentrations of IAABE (A) were 0 (■), 1.0 (♦), 2.5 (▲), 5.0 (□) and 10 µM (○). Concentrations of BAABE (B) were 0 (■), 10 (♦), 20 (▲), 40 (□) and 80 µM (○).

Figure 3

Covalent modification of tubulin by IAABE. (A) Tubulin or BSA were incubated in the presence of ³H-IAABE for 0 (▲ tubulin) or 60 minutes (■ tubulin; ♦ BSA). Samples were separated on a polyacrylamide gel and blotted onto a nitrocellulose membrane. Lanes were dissected into slices and counted for radioactivity. Tubulin has a molecular weight of 55 kDa and BSA 68 kDa. The peak at 55 kDa is monomeric tubulin and the peak at 120 kDa represents tubulin dimers. (B) CEM cells were incubated in the presence of 0.37 µM ³H-IAABE for 0 (•), 1 (■), 4 (▲) and 12 hours (♦). Cells were harvested and lysed directly into gel loading buffer. Samples were separated on a polyacrylamide gel and blotted onto a nitrocellulose membrane. Lanes were dissected into slices and counted for radioactivity. Note major peak at 55 kDa. (C) DEAE purification of ³H-IAABE-labeled tubulin from CEM cells. Extracts from ³H-IAABE-treated cells (1 hour incubation) were purified with the DEAE method. Bound tubulin (♦) and unbound fractions (■) were blotted and counted as described above. Note that 95% of radioactivity co-eluted with the tubulin.

Figure 4

Cell cycle arrest at G1/S transition in CEM cells treated with IAABE. CEM cells were seeded at 2x10⁵/ml cells/ml in the presence of IAABE at the ID₉₀ concentration 0.37 µM. After 0, 1, 4, 12, 24 and 48 hours, samples were harvested and stained for DNA content (see Methods section) before FACS analysis. Experiments were performed twice with similar results. Approximately 1000 to 5000 cells were counted per sample. Note the peak at 2.3n DNA (arrow) in the IAABE-treated cells only.

Figure 5

Interferences with the formation of microtubule structure in IAABE-treated CEM cells. All cells were treated with ID₉₀ concentration of compounds for 12 hours [12,13]. (a) Untreated control cells, (b) IAABE, (c) paclitaxel and (d) vinblastine. Insert is a DNA extracted from the same samples run on 1% agarose gels and stained with ethidium bromide. Note absence of microtubule structure and appearance of apoptotic nuclei in IAABE-treated cells. Note strong signal of apoptotic DNA ladder in IAABE-treated cells compared to vinblastine and paclitaxel samples.

Figure 6

The mode of cell death caused by IAABE. (A) Analysis of Bcl-2 phosphorylation in CEM cells treated with IAABE, vinblastine or paclitaxel (ID₉₀ concentration of compounds) for 0, 1, 3, 6, 12 and 24 hours (A, B, C, D, E and F, respectively) [13]. Note rapid appearance of phosphorylated bcl-2 (pBcl-2) in the IAABE samples. (B) DNA synthesis inhibition determined by ³H-thymidine uptake measurements. Cells were incubated with ID₉₀ of the compound [25]. (■) Vinblastine; (▲) BAABU; (♦) IAABE. Note that IAABE-treated cells shut down DNA synthesis very soon after drug application compared to BAABU and vinblastine which have a slow linear decrease in thymidine incorporation. (C) Effect of paclitaxel and IAABE on mitochondrial permeability transition. Mitochondria were incubated at 24° C in 15 µM CaCl₂ in the presence of paclitaxel or IAABE [30]. Concentration of paclitaxel were 0 (■), 5 (•), 10 (□), 20 (△) and 40 µM (○). Concentrations of IAABE were 0 (■) and 1 mM (♦). Absorbance is inversely proportional to mitochondrial swelling.

Figure 7

Effects of TDCC on the cell cycle. CEM cells were loaded with tubulin or TDCC by pinocytosis. T=1, 4 and 12 hours for tubulin only, a, b and c, respectively. T=1, 4 and 12 hours for TDCC, d, e and f, respectively. The experiment was performed twice with similar results. Approximately 1000 to 5000 cells were counted per sample. Note the peak at 2.3n DNA (arrow) in the TDCC sample only.