Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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.
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
- Table 1.
Flow cytometric analysis of Notch-1-transfected cells
G0-G1 (%) S (%) G2-M (%) BxPC-3 CS 55.8 ± 0.7 30.6 ± 1.1 13.6 ± 0.9 NS 72.4 ± 1.4 22.1 ± 1.5 5.5 ± 0.6 CP 52.3 ± 3.5 29.2 ± 3.1 18.5 ± 0.4 NP 37.8 ± 1.2 42.4 ± 2.0 19.8 ± 3.1 HPAC CS 46.9 ± 2.4 36.3 ± 1.5 16.8 ± 1.2 NS 66.2 ± 1.8 24.7 ± 1.5 9.1 ± 1.1 CP 53.8 ± 2.0 34.4 ± 1.3 11.8 ± 2.1 NP 40.1 ± 1.9 36.8 ± 1.5 23.1 ± 1.6 PANC-1 CS 40.5 ± 2.4 33.7 ± 1.8 25.8 ± 1.6 NS 67.4 ± 3.5 15.3 ± 1.2 17.3 ± 2.8 CP 46.1 ± 2.1 35.5 ± 1.5 18.4 ± 0.9 NP 29.8 ± 2.6 54.3 ± 1.2 15.9 ± 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.
Volume 5, Issue 3
