A novel synthetic analog of 5, 8-disubstituted quinazolines blocks mitosis and induces apoptosis of tumor cells by inhibiting microtubule polymerization

Abstract

Many mitosis inhibitors are powerful anticancer drugs. Tremendous efforts have been made to identify new anti-mitosis compounds for developing more effective and less toxic anti-cancer drugs. We have identified LJK-11, a synthetic analog of 5, 8-disubstituted quinazolines, as a novel mitotic blocker. LJK-11 inhibited growth and induced apoptosis of many different types of tumor cells. It prevented mitotic spindle formation and arrested cells at early phase of mitosis. Detailed in vitro analysis demonstrated that LJK-11 inhibited microtubule polymerization. In addition, LJK-11 had synergistic effect with another microtubule inhibitor colchicine on blocking mitosis, but not with vinblastine or nocodazole. Therefore, LJK-11 represents a novel anti-microtubule structure. Understanding the function and mechanism of LJK-11 will help us to better understand the action of anti-microtubule agents and to design better anti-cancer drugs.

Figure 1. Chemical structure of LJK-11.

Figure 2. Effect of LJK-11 on the growth and death of A549 cells.

A. MTT assay of LJK-11 effects on cell growth. A549 cells were incubated with indicated concentrations of LJK-11 for 48 hours. The effect on cell growth and death was examined by MTT assay. The cell viability is expressed as a percentage of the compound-treated viable cells divided by the viable cells of the untreated control. The data are the means of triplicates ±SD. B. PARP cleavage assay of LJK-11 effects on inducing cell apoptosis. A549 cells were treated with 50 µM for indicated time periods. The cell lysates were resolved by SDS-PAGE and analyzed by Western bolt analysis using anti-PARP antibody. The cleaved PARP is indicated by the arrow. Anti-tubulin antibody was used as a protein loading control.

Figure 3. Effect of LJK-11 on the growth and death of different tumor cells.

A549, Hela, HGC-27, or MDA-MB-453 cells were incubated with the indicated concentrations of LJK-11 for 48 hours. The effect of LJK-11 on cell growth and death was evaluated by MTT assay. The cell viability is expressed as a percentage of the compound-treated viable cells divided by the viable cells of the untreated control. The data are the means of triplicates ±SD.

Figure 4. Effect of LJK-11 on tyrosine phosphorylation of signaling proteins.

A549 cells were treated with 50 µM LJK-11 for 6, 12, 24, or 36 hours. The cell lysates were resolved by SDS-PAGE and analyzed by Western bolt analysis using antibodies as indicated. Antibodies specific to the phosphorylated forms of the indicated proteins are labeled with (-P).

Figure 5. Effects of LJK-11 on cell cycle distribution.

A. Flow cytometry analysis of LJK-11-treated A549 tumor cells. A549 cells were incubated with different concentrations of LJK-11 for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. B. Percentage of cells in G2/M phase after 24 hours treatment with different concentrations of LJK-11. The data are the means of triplicates ±SD. C. Flow cytometry analysis of LJK-11-treated MDA-MB-453 tumor cells. MDA-MB-453 cells were incubated with different concentrations of LJK-11 for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. D. Percentage of cells in G2/M phase after 24 hours treatment with different concentrations of LJK-11. The data are the representative of three independent experiments.

Figure 6. Effects of LJK-11 on mitotic spindle formation.

A549 cells were incubated on glass coverslips with different reagents for 16 hours, and then fixed and stained with α-tubulin antibody to visualize microtubules (green) and with DAPI to visualize chromosomes (blue). The cells were visualized by indirect immunofluorescent microscopy. A: control cells treated with equal volume of DMSO (0.1%). B: cells treated with 100 µM LJK-11. C: cells treated with 5 nM nocodazole. D: cells treated with 100 nM colchicine. E: cells treated with 50 nM Taxol.

Figure 7. Effects of LJK-11 on tubulin structure in non-mitotic cells.

A549 cells were incubated on glass coverslips with (B) or without (A) LJK-11 for 4 hours, and then fixed and stained with α-tubulin antibody to visualize microtubules (green) and with DAPI to visualize chromosomes (blue). The cells were visualized by indirect immunofluorescent microscopy.

Figure 8. Effect of LJK-11 on tubulin polymerization.

Effects of LJK-11 (250 µM), colchicines (10 µM), nocodazole (10 µM), or Taxol (10 µM) on bovine brain tubulin polymerization were measured turbidimetrically(A). Effects of 1 µM, 5 µM, 25 µM, 100 µM, 200 µM LJK-11 on bovine brain tubulin polymerization were also measured. Changes in absorbance at 340 nm (A₃₄₀) were measured and plotted as a function of time(B).

Figure 9. Reversibility of LJK-11 treatment.

Hela cells were incubated with 50 µM LJK-11 or 20 nM colchicine for 20 hours (A) and then the compound-containing media were removed, the cells were washed with fresh media, and the cells were incubated in new compound-free media for additional 18 hours (B). The pictures were taken using a light microscope.

Figure 10. Synergistic effect of LJK-11 and colchicine on blocking mitosis.

A. Flow cytometry analysis of the effects of LJK-11 (10 µM), colchicines (20 nM), or the combination of the two on cell cycle distribution. A549 cells were incubated with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. B. Percentage of cells in G2/M phase after 24 hours treatment with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine. C. Concentration dependent G2/M arrest by treatment of colchicines or LJK-11 for 24 h. Also indicated in the figures are the percentages of G2/M arrest induced by the combination of 10 µM LJK-11 and 20 nM colchicine. Data are the means of triplicates ± SD.