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. 2010 Oct;9(10):2700-13.
doi: 10.1158/1535-7163.MCT-10-0645.

Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability

Emin Oroudjev  1 , Manu Lopus, Leslie Wilson, Charlene Audette, Carmela Provenzano, Hans Erickson, Yelena Kovtun, Ravi Chari, Mary Ann Jordan
Affiliations

Maytansinoid-antibody conjugates induce mitotic arrest by suppressing microtubule dynamic instability

Emin Oroudjev et al. Mol Cancer Ther. 2010 Oct.

Abstract

Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule dynamic instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule dynamic instability.

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Figures

Figure 1
Figure 1
Structures of maytansine, DM1 and S-methyl DM1 (For DM1: R=H, For S-methyl DM1: R=Me), DM1 immunoconjugates, DM1-SMCC-lys metabolite.
Figure 2
Figure 2
Concentration dependence for (A) inhibition of proliferation, (B) G2/M arrest and (C) mitotic arrest of MCF7 cells by maytansinoids and their antibody conjugates. Cells were incubated with (○) maytansine, (●) S-methyl DM1, (□) B38.1-SPP-DM1 and (■) B38.1-SMCC-DM1. (A) Cells were incubated with compound for 72 h and inhibition of proliferation was measured by SRB (Materials and Methods). Maytansine, S-methyl DM1, B38.1-SPP-DM1 and B38.1-SMCC-DM1 inhibited proliferation with IC50 values 710 pmol/L, 330 pmol/L, 11 nmol/L and 5.2 nmol/L, respectively. (B) Cells were incubated with compounds for 24 h and the population of G2/M cells was determined by flow cytometry (Materials and Methods). Maytansine, S-methyl DM1, B38.1-SPP-DM1 and B38.1-SMCC-DM1 induced arrest in G2/M with IC50 values 310 pmol/L, 340 p mol/L, 10 nmol/L and 6.8 nmol/L, respectively. (C) Cells were incubated with S-methyl DM1 for 24 h and the percentage of cells that stained positively for phosphorylated histone H3 was determined by flow cytometry or counted by microscopy (Materials and Methods). S-methyl DM1 induced mitotic arrest with an IC50 of 400 nmol/L. Results are means and standard error of at least three experiments.
Figure 3
Figure 3
Effects of S-methyl DM1 on microtubule organization in interphase and mitosis. (A) Interphase: MCF7 cells were fixed and stained for α-tubulin (green), actin (red) and DNA (blue). Control cells (a) and cells treated for 24 h with 340 pmol/L (mitotic IC50) S-methyl DM1 (b) show intact microtubule and actin networks with similar overall morphologies. (c) At 680 pmol/L S-methyl DM1 (2 × mitotic IC50), microtubules were significantly shorter (arrowheads), reduced in number, and showed a patchy distribution. Some tubulin aggregates are visible (arrows). At 1.4 nmol/L S-methyl DM1 (4 × mitotic IC50) (d) all microtubules were depolymerized and actin formed a narrow band at the cell periphery. Scale bar = 10 μm. (B) Mitosis: MCF7 cells were incubated for 24 h with medium alone (e) or with 340 pmol/L (1 × mitotic IC50) (f,g,h,i,j) or 680 pmol/L S-methyl DM1(2 × mitotic IC50) (k,l). Cells were fixed and stained for α-tubulin (green), the centrosome- associated protein pericentrin (red) and DNA (blue). In control mitotic cells (e), spindles were normal with well-separated centrosomes and with the chromosomes congressed to a compact metaphase plate. The majority of cells arrested in mitosis by S-methyl DM1 displayed some abnormalities in metaphase: uncongressed chromosomes (red arrows), poorly-defined bipolar spindles or monopolar spindles, an increased number of astral microtubules, and poorly-separated centrosomes (white arrows). Based on these morphological abnormalities cells were categorized into Type I, Type II, Type III and Type IV spindles (see Results). Scale bar = 5 μm.
Figure 4
Figure 4
Comparison of the cellular accumulation of S-[3H]methyl-DM1 with accumulation of the metabolites of cleavable and uncleavable B38.1-SPP-[3H]DM1 conjugates. (A) Time course of uptake of 780 pmol/L [3H]-S-methyl DM1 by MCF7 cells. (B) Radio chromatograms of the [3H] maytansinoid metabolites of B38.1-SPP-[3H]DM1 and B38.1-SMCC-[3H]DM1. MCF-7 cells were exposed to the B38.1-SPP-[3H]DM1 conjugates for the indicated of the homogenates were assayed for 3H-maytansinoid metabolites by extraction and HPLC. The effluent from the samples was collected in 1 mL fractions and the radioactivity corresponding to each fraction was determined by LSC. The counts per minute of tritium (CPM) associated with the fractions was plotted vs. the time the fraction was collected. . The lowest two panels show that co-incubation with 1 μmol/L unconjugated EpCAM antibody strongly inhibited the formation of the metabolites. (C) Diagram depicting the cellular uptake and processing of the anti-EpCAM antibody-DM1 conjugates, B38.1-SMCC-DM1 and B38.1-SPP-DM1. Both conjugates are internalized by antigen-mediated endocytotosis. The endosomes containing conjugate fuse with lysosomes, and the conjugates undergo proteolysis forming the corresponding lysine-linker-maytansinoids, lysine-SMCC-DM1 and lysine-SPP-DM1. The lysine-SPP-DM1 metabolite of the cleavable conjugate is further reduced to yield free DM1 while the lysine-SMCC-DM1 metabolite of the uncleavable conjugate resists additional processing.
Figure 5
Figure 5
Dynamic instability of microtubules in MCF7 cells incubated with maytansine, S-methyl DM1, B38.1-SMCC-DM1, and B38.1-SPP-DM1. Cells were incubated for 5 h with 310 pmol/L maytansine, 340 pmol/L S-methyl DM1, 10.5 nmol/L B38.1-SPP-DM1 and 6.8 nmol/L B38.1-SMCC-DM1 (1×IC50 concentrations of corresponding compounds) or medium alone. Time-lapse images (A) of individual microtubules in the lamellar periphery of cells were recorded by epifluorescence microscopy. Microtubule ends (black triangles) were tracked over time to produce life history plots. (B) Life history plots of length of individual microtubules. In all cells treated with maytansinoids the dynamic instability of individual microtubules was visibly suppressed. (C and D) Over the 24-h time course of incubation with B38.1-SMCC-DM1 (C) and B38.1-SPP-DM1 (D) suppression of microtubule dynamicity (left axes) increased similarly with the increase in the intracellular concentration of the metabolite of the B38.1-DM1 conjugates as determined by the AUC (area under the curve)(right axes) as shown in Fig. 4C.
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