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Molecular Cancer Therapeutics
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Article

Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells

Zhiwei Wang, Yuxiang Zhang, Yiwei Li, Sanjeev Banerjee, Joshua Liao and Fazlul H. Sarkar
Zhiwei Wang
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Yuxiang Zhang
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Yiwei Li
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Sanjeev Banerjee
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Joshua Liao
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Fazlul H. Sarkar
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DOI: 10.1158/1535-7163.MCT-05-0299 Published March 2006
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    Figure 1.

    Constitutive expression and down-regulation of Notch-1 by siRNA in pancreatic cancer cell lines BxPC-3, HPAC, and PANC-1. CS, control siRNA; NS, Notch-1 siRNA; CP, control plasmid; NP, Notch-1 plasmid. A and B, Notch-1 mRNA and protein levels were measured by real-time RT-PCR and Western blotting. C, Notch-1 mRNA levels were down-regulated by Notch-1 siRNA. D, top, Notch-1 protein levels were down-regulated by siRNA in all three pancreatic cancer cells. Bottom, Notch-1 protein levels were overexpressed by cDNA transfection in different cell lines.

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    Figure 2.

    Effect of down-regulation of Notch-1 by siRNA on pancreatic cancer cell growth and apoptosis. A to C, inhibition of cancer cell growth tested by MTT assay. D to F, cell death assay for measuring apoptosis tested by ELISA. *, P < 0.05; **, P < 0.01, relative to control siRNA.

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    Figure 3.

    Effect of up-regulation of Notch-1 by cDNA plasmid on pancreatic cancer cell growth and apoptosis. A to C, promotion of cancer cell growth tested by MTT assay. D to F, cell death assay for measuring apoptosis tested by ELISA. *, P < 0.05; **, P < 0.01, relative to control plasmid.

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    Figure 4.

    Effect of the down-regulation of Notch-1 on cell cycle distribution. HPAC cells were harvested for cell cycle analysis using propidium iodide staining. X axis, DNA content; Y axis, number of nuclei. Compared with the control, down-regulation of Notch-1 caused G0-G1 cell cycle arrest.

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    Figure 5.

    The level of expression of several known G0-G1 cell cycle regulatory factors as detected by Western blotting in all three pancreatic cancer cells.

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    Figure 6.

    Down-Regulation of Notch-1 gene expression by Notch-1 siRNA inhibited NF-κB DNA-binding activity. Nuclear proteins from siRNA- and cDNA-transfected cells were subjected to analysis for NF-κB DNA-binding activity as measured by EMSA. A to C, down-regulation of Notch-1 inhibited NF-κB DNA-binding activity compared with control whereas Notch-1 cDNA transfection caused activation of NF-κB DNA-binding activity in all three cell lines tested. D, NF-κB supershift analyses. EMSA experiments were done by additional 30-min incubations with polyclonal supershift antibodies against p65 before the addition of labeled probe. 1, nonspecific antibody (anti–cyclin D1); 2, p65 antibody.

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    Figure 7.

    Down-regulation of Notch-1 by genistein inhibited cell growth and induced apoptosis. A, inhibition of Notch-1 mRNA after 6, 12, 18, 24, 48, and 72 h of treatment with 25 μmol/L genistein in BxPC-3 pancreatic cancer cells. The mRNA level in BxPC-3 pancreatic cancer cells treated with genistein was assessed by real-time RT-PCR. The expression of Notch-1 at the mRNA level was down-regulated after genistein treatment. *, P < 0.05; * *, P < 0.01, relative to solvent control. B, inhibition of Notch-1, Hes-1, cyclin D1, and Bcl-XL protein expression by 25 μmol/L genistein in BxPC-3 pancreatic cancer cells. Cells were treated with 25 μmol/L genistein for 24, 48, and 72 h. Western blot analysis showed that the protein levels of Notch-1, Hes-1, Bcl-XL, and cyclin D1 were down-regulated in genistein-treated BxPC-3 pancreatic cancer cells in a time-dependent manner. C, inhibitory effect of genistein on the growth of BxPC-3 pancreatic cancer cells tested by MTT assay. BxPC-3 cells treated with 15, 25, and 50 μmol/L genistein for 3 d. The treatment of BxPC-3 pancreatic cancer cells with genistein resulted in cell growth inhibition. The inhibition of cell growth was dose and time dependent. *, P < 0.01, relative to solvent control. D, genistein-induced apoptosis in BxPC-3 pancreatic cancer cells measured by the histone/DNA fragment analysis using ELISA. Cells were treated with 25 μmol/L genistein for 24, 48, or 72 h. The induction of apoptosis was time dependent and was found to be more pronounced after 48 to 72 h of treatment. *, P < 0.01, relative to solvent control. E, NF-κB DNA binding activity was measured by EMSA. Genistein inhibits NF-κB DNA binding activity in BxPC-3 pancreatic cancer cells. Cells were treated with 10, 25, and 50 μmol/L genistein for 48 h. Nuclear extracts were prepared from control and genistein-treated cells and subjected to analysis for NF-κB DNA binding activity as measured by EMSA. Genistein significantly inhibited NF-κB DNA-binding activity whereas there was no change in Rb (used as a protein loading control).

  • Figure 8.
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    Figure 8.

    BxPC-3 pancreatic cancer cell growth inhibition and cell death induced by Notch-1 siRNA and genistein. A, Notch-1 expression was down-regulated by genistein and Notch-1 siRNA. Western blot analysis was used to detect the protein level of Notch-1. 1, control; 2, 25 μmol/L genistein; 3, Notch-1 siRNA; and 4, Notch-1 siRNA plus 25 μmol/L genistein. B, down-regulation of Notch-1 expression significantly inhibited cell growth. Genistein plus siRNA Notch-1 inhibited cell growth to a greater degree compared with genistein alone. C, BxPC-3 pancreatic cancer cell death induced by Notch-1 siRNA and genistein. Down-regulation of Notch-1 expression significantly increased apoptosis induced by genistein. Notch-1 siRNA–transfected BxPC-3 cells were significantly more sensitive to spontaneous and genistein-induced apoptosis. D, NF-κB DNA binding activity was measured by EMSA. Genistein and Notch-1 siRNA significantly inhibited NF-κB DNA-binding activity. Genistein plus Notch-1 siRNA inhibited NF-κB DNA binding activity to a greater degree compared with genistein alone. Rb level served as nuclear protein loading control. Control, cells treated with DMSO; 1, control; 2, 25 μmol/L genistein; 3, Notch-1 siRNA; and 4, Notch-1 siRNA plus 25 μmol/L genistein.

  • Figure 9.
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    Figure 9.

    Overexpression of Notch-1 by cDNA transfection reduced genistein-induced cell growth inhibition and apoptosis. A, the efficacy of Notch-1 cDNA for overexpression of Notch-1 protein was tested by Western blot analysis. B and C, overexpression of Notch-1 by cDNA transfection rescued genistein-induced cell growth inhibition and abrogated genistein-induced apoptosis to a certain degree. D, overexpression of Notch-1 by cDNA transfection partly abrogated inactivation of NF-κB DNA-binding activity by genistein in BxPC-3 cells whereas there was no change in the levels of Rb (used as protein loading control). Control, cells treated with DMSO; 1, control plasmid; 2, control plasmid plus 25 μmol/L genistein; 3, Notch-1 cDNA; and 4, Notch-1 cDNA plus 25 μmol/L genistein.

Tables

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  • Table 1.

    Flow cytometric analysis of Notch-1-transfected cells

    G0-G1 (%)S (%)G2-M (%)
    BxPC-3
        CS55.8 ± 0.730.6 ± 1.113.6 ± 0.9
        NS72.4 ± 1.422.1 ± 1.55.5 ± 0.6
        CP52.3 ± 3.529.2 ± 3.118.5 ± 0.4
        NP37.8 ± 1.242.4 ± 2.019.8 ± 3.1
    HPAC
        CS46.9 ± 2.436.3 ± 1.516.8 ± 1.2
        NS66.2 ± 1.824.7 ± 1.59.1 ± 1.1
        CP53.8 ± 2.034.4 ± 1.311.8 ± 2.1
        NP40.1 ± 1.936.8 ± 1.523.1 ± 1.6
    PANC-1
        CS40.5 ± 2.433.7 ± 1.825.8 ± 1.6
        NS67.4 ± 3.515.3 ± 1.217.3 ± 2.8
        CP46.1 ± 2.135.5 ± 1.518.4 ± 0.9
        NP29.8 ± 2.654.3 ± 1.215.9 ± 1.7
    • NOTE: Flow cytometry was done as described in Fig. 4. The mean values (± SE) represent the percentage of cells in the indicated phase of the cell cycle from three independent experiments.

      Abbreviations: CS, control siRNA; NS, Notch-1 siRNA; CP, control plasmid; NP, Notch-1 plasmid.

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Molecular Cancer Therapeutics: 5 (3)
March 2006
Volume 5, Issue 3
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Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells
Zhiwei Wang, Yuxiang Zhang, Yiwei Li, Sanjeev Banerjee, Joshua Liao and Fazlul H. Sarkar
Mol Cancer Ther March 1 2006 (5) (3) 483-493; DOI: 10.1158/1535-7163.MCT-05-0299

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Down-regulation of Notch-1 contributes to cell growth inhibition and apoptosis in pancreatic cancer cells
Zhiwei Wang, Yuxiang Zhang, Yiwei Li, Sanjeev Banerjee, Joshua Liao and Fazlul H. Sarkar
Mol Cancer Ther March 1 2006 (5) (3) 483-493; DOI: 10.1158/1535-7163.MCT-05-0299
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