Reversibility of LJK-11 treatment.

<p>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.</p

An insight into the curdione biotransformation pathway by <i>Aspergillus niger</i>

<div><p>Curdione (<b>1</b>), a sesquiterpene with a germacrane skeleton from rhizomes of <i>Curcuma wenyujin</i>, has attracted attention due to its important pharmacological properties. Herein, we investigated the chemo-biotransformation of curdione (<b>1</b>) systematically using <i>Aspergillus niger</i> AS 3.739. Regio- and stereoselective hydroxylation of curdione with filamentous fungus <i>A. niger</i> AS 3.739 led to seven metabolites including four new compounds 3α-hydroxycurcumalactone, 2β-hydroxycurcumalactone, (10<i>S</i>)-9,10-dihydroxy-curcumalactone and (10<i>R</i>)-9,10-dihydroxy-curcumalactone. Their structures were determined by spectroscopic techniques including two-dimensional NMR and TOF-MS (Time of Flight Mass Spectrometry). Based upon the analysis of biological and chemical conversions of curdione, a tentative metabolic pathway via chemo-bio cascade reactions is proposed in <i>A. niger</i> system, which provides an insight into the corresponding metabolism of curdione in animal systems. In addition, experiments with selected monooxygenase inhibitors suggest that cytochrome P450 monooxygenase played a crucial role in the hydroxylation of curdione.</p></div

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

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

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 the growth and death of different tumor cells.

<p>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.</p

Mitaplatin Increases Sensitivity of Tumor Cells to Cisplatin by Inducing Mitochondrial Dysfunction

Tumor resistance to chemotherapy is the major obstacle to employ cisplatin, one of the broadly used chemotherapeutic drugs, for effective treatment of various tumors in the clinic. Most acknowledged mechanisms of cancer resistance to cisplatin focus on increased nuclear DNA repair or detoxicity of cisplatin. We previously demonstrated that there was a unique metabolic profile in cisplatin-resistant (CP-r) human epidermoid adenocarcinoma KB-CP 20 and hepatoma BEL 7404-CP 20 cancer cells. In this study, we further defined hyperpolarized mitochondrial membrane potentials (Δψ_m) in CP-r KB-CP 20 and BEL 7404-CP 20 cells compared to the cisplatin-sensitive (CP-s) KB-3-1 and BEL 7404 cells. Based on the mitochondrial dysfunction, mitaplatin was designed with two mitochondrial-targeting moieties [dichloroacetate (DCA) units] to the axial positions of a six-coordinate Pt­(IV) center to sensitize cisplatin resistance. It was found that mitaplatin induced more apoptosis in CP-r KB-CP 20 and BEL 7404-CP 20 cells than that of cisplatin, DCA and cisplatin/DCA compared on an equal molar basis. There was more platinum accumulation in mitaplatin-treated CP-r cells due to enhanced transmembrane permeability of lipophilicity, and mitaplatin also showed special targeting to mitochondria. Moreover, in the case of treatment with mitaplatin, the dramatic collapse of Δψ_m was shown in a dose-dependent manner, which was confirmed by FACS and confocal microscopic measurements. Reduced glucose utilization of CP-r cells was detected with specifically inhibited phosphorylation of pyruvate dehydrogenase (PDH) at Ser-232, Ser-293, and Ser-300 of the E1α subunit when treated with mitaplatin, which was indicated to modulate the abnormal glycolysis of resistant cells. The present study suggested novel mitochondrial mechanism of mitaplatin circumventing cisplatin resistance toward CP-r cells as a carrier across membrane to produce CP-like cytotoxicity and DCA-like mitochondria-dependent apoptosis. Therefore, mitochondria targeting compounds would be more vulnerable and selective to overcome cisplatin resistance due to the unique metabolic properties of CP-r cancer cells