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Molecular Cancer Therapeutics
Molecular Cancer Therapeutics

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CP248, a Derivative of Exisulind, Causes Growth Inhibition, Mitotic Arrest, and Abnormalities in Microtubule Polymerization in Glioma Cells 1 Supported by awards from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Cancer Research (to I. B. W.). Additional grant support was from the American Cancer Society and NIH (to G. G. G.). A. F. P. was supported by a fellowship from the Fonds de la Recherche en Santé du Québec.1

Jung-Taek Yoon, Alexander F. Palazzo, Danhua Xiao, Thomas M. Delohery, Peter E. Warburton, Jeffrey N. Bruce, W. Joseph Thompson, Gerhard Sperl, Clark Whitehead, John Fetter, Rifat Pamukcu, Gregg G. Gundersen and I. Bernard Weinstein
Jung-Taek Yoon
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Alexander F. Palazzo
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Danhua Xiao
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Thomas M. Delohery
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Peter E. Warburton
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Jeffrey N. Bruce
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W. Joseph Thompson
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Gerhard Sperl
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Clark Whitehead
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John Fetter
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Rifat Pamukcu
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Gregg G. Gundersen
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I. Bernard Weinstein
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DOI:  Published April 2002
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  • Fig 1.
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    Fig 1.

    Chemical structures of exisulind, CP461, CP248, and colchicine.

  • Fig 2.
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    Fig 2.

    Effects of CP248 on mitotic 9L cells. A, a mitotic 9L cell after treatment with 0.1% DMSO (solvent control). B, a 9L cell treated with 50 ng/ml Colcemid (positive control). C, a mitotic 9L cell after treatment with 0.075 μm CP248. Cells were treated for 12 h and fixed with methanol. Chromosomal DNA was stained blue with DAPI. MTs were stained green and MAD2 proteins were stained red with the respective antibodies and were visualized by indirect immunofluorescence, as described in “Materials and Methods.” Abnormal spindle apparatus is notable in the CP248-treated cells. White bar, 10 μm.

  • Fig 3.
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    Fig 3.

    Effects of CP248 on interphase 9L cells. The 9L cells were treated with 0.075 μm CP248 (A, C) or 0.1% DMSO (B, D) for 6 h, fixed with methanol; and DNA, MTs, and centrosomes were visualized by indirect immunofluorescence, as described in Fig. 2 and “Materials and Methods.” In B, DMSO-treated 9L cells show a tight correlation of centrosomes (stained red) with the MT array (white arrow). In A, CP248-treated 9L cells show a dissociation of the centrosome (white arrow) from the MT array (yellow arrow). In C, CP248-treated 9L cells were stained for stable Glu MTs (red) and DNA (blue). In D, DMSO-treated 9L cells were stained for stable Glu MT (red) and for DNA (blue). White bars, 20 μm.

  • Fig 4.
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    Fig 4.

    Effects of CP248 on MTs in interphase 9L cells. 9L cells were treated with 0.1% DMSO (A), 0.2 μm CP248 (B), or 0.4 μm CP248 (C) for 2 h. The cells were fixed with methanol, and MTs were visualized by immunofluorescence, as described in “Materials and Methods.” The 9L cells were treated with 0.4 μm CP248 for 2 h, washed three times with fresh medium, and then incubated for an additional 15 (D), 30 (E), or 60 (F) min, before fixation and immunostaining. White bars, 20 μm.

  • Fig 5.
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    Fig 5.

    Effects of CP248 on MTs in NIH 3T3 fibroblasts. The cells were grown in medium in the absence of serum for 48 h. They were then incubated in medium minus serum containing: 0.1% DMSO (A); 0.1 μm CP248 (B); 200 μm exisulind (C); 1.5 μm CP461 (D); 0.1 μm CP248 plus 10 μm KT5823 (E); 0.1 μm CP248 plus 10 μm Rpt-8-pCPT0cGMP (F); 170 nm YC-1 (G); or 4 mm dibutyryl cGMP (H). After 2 h of incubation with these agents, the cells were fixed with methanol and the MTs visualized by immunofluorescence, as described in “Materials and Methods.” White bars, 20 μm.

  • Fig 6.
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    Fig 6.

    Effects of CP248 on in vitro MT polymerization, measured by turbidity. A solution of MTs at 4°C was placed into a thermostable chamber of a spectrophotometer and incubated at 37°C. Absorbance at 350 nm was then determined during the indicated time course. After MT polymerization reached a steady state (20 min), 5 μm Taxol, 0.1 μm CP248, 1 μm CP248, or 1.35 μm Colcemid were added. Because Colcemid and CP248 gave some absorbance at 350 nm, this value was subtracted from the total absorbance. For additional details, see “Materials and Methods.”

Tables

  • Figures
  • Table 1.

    Inhibition of cell proliferation by sulindac sulfone (exisulind) and derivatives in a series of glioma cell lines

    Cell proliferation was determined by tritiated thymidine incorporation, as described in “Materials and Methods.” IC50 values, calculated from a concentration curve, indicate the concentrations of the drugs that caused a 50% inhibition of tritiated thymidine incorporation when compared with a control culture treated only with the vehicle, 0.1% DMSO. Data are the mean values from two separate experiments in which all of the assays were done in triplicate. Repeat assays differed by less than 10%.

    Cell lineIC50 (μm)
    ExisulindCP461CP248
    Rat glioma cell lines
     9L1791.140.078
     C61681.080.068
     F98960.7250.075
     D741371.020.064
    Human glioma cell lines
     U871401.430.083
     U1381401.190.070
     U3731450.980.077
     A1721351.170.078
     GB12400.80.069
     GB21801.350.073
     GB31721.040.067
     GB41451.090.088
    NIH mouse fibroblasts
     3T32001.50.108
  • Table 2.

    Flow cytometric analysis

    Cell cycle analysis was performed as described in “Materials and Methods.” Cells were treated with the indicated compounds at their respective IC50 values (see Table 1), collected at the indicated time points, and analyzed by DNA flow cytometry. The values obtained are an average of two independent experiments, with a minimum of 15,000 events collected for each time point. Duplicates agreed within 5%.

    Treatment
    6 h (%)
    12 h (%)
    24 h (%)
    9L cell line
    No treatment
     G0-G146.148.452.7
     S32.736.132.0
     G219.313.813.8
     M1.81.81.6
     >G2-M3.92.93.5
    CP248 (0.078 μm)
     G0-G127.231.832.8
     S35.228.442.4
     G224.524.220.5
     M13.115.74.4
     >G2-M4.07.017.4
    CP461 (1.14 μm)
     G0-G144.444.655.4
     S33.536.930.0
     G219.715.912.7
     M2.42.71.9
     >G2-M2.62.72.3
    Exisulind (180 μm)
     G0-G148.850.758.4
     S30.234.528.8
     G219.513.211.5
     M1.61.71.3
     >G2-M2.12.32.4
    GB1 cell line
    No treatment
     G0-G147.554.738.0
     S17.813.930.9
     G232.729.228.7
     M2.02.22.4
     >G2-M8.76.49.4
    CP248 (0.07 μm)
     G0-G144.142.521.1
     S18.717.330.4
     G232.930.332.1
     M4.99.916.4
     >G2-M8.58.811.7
    CP461 (0.8 μm)
     G0-G148.754.238.1
     S19.014.730.2
     G229.127.528.3
     M2.33.73.4
     >G2-M7.56.59.7
    Exisulind (24 μm)
     G0-G147.953.749.0
     S19.214.124.8
     G231.229.824.3
     M1.82.42.0
     >G2-M7.87.47.1
    Colcemid (0.13 nm)
     G0-G141.642.411.8
     S19.713.842.0
     G237.138.435.8
     M1.73.910.4
     >G2-M9.910.113.6
  • Table 3.

    MAD2 expression

    The human glioma cell line GB2 was treated with CP248, CP461, or exisulind at the respective IC50 concentrations for 18 h and immunostained for MAD2, as described in “Materials and Methods.” MAD2 overexpression was defined as the percentage of mitotic cells in which >50% of the chromosomes stained positive for MAD2. An example is shown in Fig. 2C. Mitotic cells on a single glass coverslip were scored for MAD2 expression by microscopy with a ×100 Nikon objective. More than 150 mitotic cells were scored for each of the treatment conditions.

    DrugGB2 Mad2 overexpression
    (%)(SD)
    No drug180
    Colcemid982.4
    CP248585.7
    CP461201.4
    Exisulind181.4
  • Table 4.

    Effects of the IC50 concentration of CP248 on stabilized MT in 9L cells

    The 9L cells were treated with the IC50 concentration of CP248 for 6 h, fixed, and stained for stable MT with the SG antibody, as described in “Materials and Methods” and Figs. 3, C and D. A positive cell was defined as any cell that expressed 12 or more SG-stained MT or very bright foci of SG. Bright foci of SG represent a large amount of stabilized MT in a concentrated region of the cell. This experiment was repeated twice with similar results. A minimum of 150 cells were scored in each experiment.

    Drug% of cells positiveSD
    None21.85±0.9
    CP24843.85±10.4
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Molecular Cancer Therapeutics: 1 (6)
April 2002
Volume 1, Issue 6
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CP248, a Derivative of Exisulind, Causes Growth Inhibition, Mitotic Arrest, and Abnormalities in Microtubule Polymerization in Glioma Cells 1 Supported by awards from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Canc…
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CP248, a Derivative of Exisulind, Causes Growth Inhibition, Mitotic Arrest, and Abnormalities in Microtubule Polymerization in Glioma Cells 1 Supported by awards from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Cancer Research (to I. B. W.). Additional grant support was from the American Cancer Society and NIH (to G. G. G.). A. F. P. was supported by a fellowship from the Fonds de la Recherche en Santé du Québec.1
Jung-Taek Yoon, Alexander F. Palazzo, Danhua Xiao, Thomas M. Delohery, Peter E. Warburton, Jeffrey N. Bruce, W. Joseph Thompson, Gerhard Sperl, Clark Whitehead, John Fetter, Rifat Pamukcu, Gregg G. Gundersen and I. Bernard Weinstein
Mol Cancer Ther April 1 2002 (1) (6) 393-404;

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CP248, a Derivative of Exisulind, Causes Growth Inhibition, Mitotic Arrest, and Abnormalities in Microtubule Polymerization in Glioma Cells 1 Supported by awards from Cell Pathways, Inc., the T. J. Martell Foundation, and the National Foundation for Cancer Research (to I. B. W.). Additional grant support was from the American Cancer Society and NIH (to G. G. G.). A. F. P. was supported by a fellowship from the Fonds de la Recherche en Santé du Québec.1
Jung-Taek Yoon, Alexander F. Palazzo, Danhua Xiao, Thomas M. Delohery, Peter E. Warburton, Jeffrey N. Bruce, W. Joseph Thompson, Gerhard Sperl, Clark Whitehead, John Fetter, Rifat Pamukcu, Gregg G. Gundersen and I. Bernard Weinstein
Mol Cancer Ther April 1 2002 (1) (6) 393-404;
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