A Novel Synthetic Analog of 5, 8-Disubstituted Quinazolines Blocks Mitosis and Induces Apoptosis of Tumor Cells by Inhibiting Microtubule Polymerization

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

MOESM1 of Cyclic helix B peptide inhibits ischemia reperfusion-induced renal fibrosis via the PI3K/Akt/FoxO3a pathway

Additional file 1: Figure S1. Validation of FoxO3a siRNA and Wortmannin. The p-FoxO3a proteins (A) and mRNA (B) expression of HK-2 cells were significantly down-regulated by FoxO3a shRNA treatment. The expression of p-Akt was also significantly reduced by Wortmannin in the HK-2 cells (C)

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

<p>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).</p

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

<p><b>A</b>. 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>B</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. <b>C</b>. 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.</p

Effects of LJK-11 on mitotic spindle formation.

<p>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.</p

Chemical structure of LJK-11.

<p>Chemical structure of LJK-11.</p

Effect of LJK-11 on tubulin polymerization.

<p>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).</p

Effects of LJK-11 on cell cycle distribution.

<p>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.</p