Article Figures & Data
Figures
- Figure 1.
Reduced tube formation of HUVECs induced by conditioned media from LNCaP and C4-2B cells treated with B-DIM. Growth factor–reduced Matrigel (125 μL) after being thawed on ice was plated in an eight-well chamber. The chamber was then incubated at 37°C for 30 min to allow the Matrigel to polymerize. HUVECs (5 × 104 cells per well) were seeded into each well in conditioned medium. A, conditioned medium was from LNCaP and C4-2B cells treated with either DMSO (CM) or treated with 10 μmol/L B-DIM (CMB) in serum-free medium for 24 h and conditioned medium from LNCaP and C4-2B cells preincubated with 10 μg/mL VEGF neutralizing antibody at 37°C for 1 h (CMAV). The chamber was incubated for 6 h. The well was photographed using an inverted microscope with a digital camera. Results are representative of three independent experiments. B, image analysis of tubule/capillary length was carried out using software image analysis program Scion Image downloaded from NIH website. Quantification of cumulative tube length of endothelial cells as observed in (A). Columns, mean; bars, SE. n = 4. *, P < 0.05; **, P < 0.01 compared with conditioned media control. C, medium only was used as negative control; 50 ng/mL VEGF was used as positive control. To rule out the possibility that inhibition of tube formation by conditioned media from LNCaP and C4-2B cells treated with 10 μmol/L of B-DIM was mediated through direct effect of B-DIM on tube formation of HUVECs within 6 h incubation, HUVECs were cultured in RPMI 1640 containing DMSO or 10 μmol/L of B-DIM. The chamber was incubated for 6 h. The well was photographed using an inverted microscope with a digital camera. Results are representative of three independent experiments. D, image analysis of tubule/capillary length was carried out using software image analysis program Scion Image downloaded from NIH website. Quantification of cumulative tube length of endothelial cells cultured in medium alone or 50 ng/mL VEGF (top) and treated with DMSO or 10 μmol/L B-DIM (bottom). Columns, mean; bars, SE. n = 4. **, P < 0.01 compared with medium alone.
- Figure 2.
The effects of B-DIM on the secretion and expression of cellular VEGF. LNCaP (A) and C4-2B (B) cells were seeded at a density of 2 × 105 per well in a six-well plate. After 24 h, the cells were incubated with serum-free medium for another 24 h. The cells were then treated with 1 and 10 μmol/L of B-DIM or DMSO as vehicle control in 1% FBS for 24 h. The culture media was collected, and VEGF was assayed using an ELISA kit. Results were normalized to the cell number and expressed as pg/mL/105 cells. Columns, mean; bars, SE. n = 6. *, P < 0.05; **, P < 0.01 compared with control. C, cell culture conditions same as above, the cell lysates were subjected to gel electrophoresis and Western blot analysis was done using anti-VEGF antibody. β-Actin protein was used as loading control. D, quantitative analysis of VEGF expression in LNCaP and C4-2B cells was done and relative density of bands normalized for β-actin (control was assigned the value of 100%). Columns, mean; bars, SE. n = 3. *, P < 0.05 compared with control.
- Figure 3.
The effects of B-DIM on VEGF mRNA and promoter activity and the relative distribution of mRNA levels of VEGF isoforms. A, the relative distribution of mRNA levels of VEGF isoforms in LNCaP and C4-2B cells, respectively. B, LNCaP and C4-2B cells grown in 1% FBS were treated with 10 μmol/L of B-DIM or DMSO as control for 24 h. The total RNA was isolated using the Trizol reagent. Real-time RT-PCR was done, and specific primers and probes for total VEGF, VEGF121, VEGF165, and VEGF189 were used to quantify mRNA levels. β-Actin was used for internal control to correct the potential variation in RNA loading. *, P < 0.05. n = 4. C, pGL3-basic luciferase reporter or pGL3-2274 containing the VEGF promoter, enhancer, and firefly luciferase reporter gene were cotransfected with CMV-β-galactosidase plasmid into LNCaP and C4-2B cells. After 24 h of transfection, the cells were treated with 10 μmol/L of B-DIM or DMSO as vehicle control for another 24 h. Luciferase activity was determined using Steady-Glo Luciferase assay system (Promega). β-Galactosidase activity was used as a control for transfection efficiency. Fold increase in luciferase activity was calculated relative to the luciferase activity of pGL3-basic assigned the value of 1. B-DIM (10 μmol/L) treatment decreased the VEGF promoter activity in LNCaP but not in C4-2B cells. *, P < 0.05 compared with without B-DIM control. n = 6.
- Figure 4.
B-DIM repressed secretion and expression of MMP-9 and regulation of VEGF release by MMP-9. LNCaP (A)and C4-2B (B) cells were seeded at a density of 2 × 105 per well in six-well plate. After 24 h, the cells were cultured in serum-free medium and incubated for another 24 h, and then treated with B-DIM or DMSO as vehicle control in 1% FBS for 24 h. The culture media were collected and concentrated using the Microcon concentrator. MMP-9 levels were assayed using ELISA kit. The results were normalized to the cell number. The cell lysates were subjected to Western blotting using anti–MMP-9 antibody. β-Actin protein was used as loading control. *, P < 0.05; **, P < 0.01 compared with without B-DIM control. LNCaP (C) and C4-2B (D) cells were transfected with MMP-9 plasmid or pcDNA3 control plasmid using the effectene transfection reagent. After 18 h transfection, cells were treated with 10 μmol/L of B-DIM in serum-free media and incubated for 24 h. The culture media were collected for VEGF assay. Cell lysates were subjected to Western blotting using anti–MMP-9 antibody. β-Actin protein was used as loading control. *, P < 0.05; **, P < 0.01 compared with pcDNA control; #, P < 0.01 compared with MMP-9 group.
- Figure 5.
uPA regulated the release of VEGF and B-DIM repressed uPA expression and NF-κB DNA binding activity. A, LNCaP and C4-2B cells were transfected with uPA siRNA or control siRNA. The media were removed after 18 h transfection, and the cells were incubated in serum-free media for 24 h. The culture media were collected, centrifuged to remove cellular debris, and stored at −70°C until assay for VEGF. The cells were counted and cell lysates were subjected to gel electrophoresis, and Western blotting was done using anti-uPA antibody. B, LNCaP and C4-2B cells were seeded at a density of 2 × 105 per well in six-well plate. After 24 h, the cells were cultured in serum-free medium and incubated for another 24 h, and then treated with B-DIM or DMSO as vehicle control in 1% FBS for 24 h. Cell lysates from LNCaP and C4-2B cells were subjected to gel electrophoresis, and Western blotting was done using anti-uPA antibody. β-Actin protein was used as loading control. C, EMSA was done by incubating 3 μg of nuclear protein extracts from LNCaP and C4-2B cells treated with 10 μmol/L B-DIM or DMSO as vehicle control in 1% FBS for 24 h with IRDye 700–labeled NF-κB oligonucleotide. Retinoblastoma protein was used as a loading control. **, P < 0.01 compared with control siRNA. n = 4.
- Figure 6.
B-DIM inhibited LNCaP and C4-2B cell invasion and in vivo angiogenesis. The effects of B-DIM treatment on cell invasion were determined by using BD BioCoat Tumor Invasion Assay System. LNCaP (A) and C4-2B (B) cells with serum-free media containing 10 and 25 μmol/L of B-DIM or DMSO were seeded into the upper chamber of the system. Bottom wells were filled with complete media. After 24 h incubation, the cells invaded through the Matrigel membrane were stained with 4 μg/mL Calcein AM. The fluorescently labeled cells were photographed under a fluorescence microscope. The fluorescence of the invaded cells was read at excitation/emission wavelengths of 530/590 nm. C, C4-2B cells (4 × 106 in 50 μL PBS admixed with 0.25 mL Matrigel) were injected s.c. into the bilateral flanks of each mouse. The control group of mice was gavaged with the vehicle (sesame oil), whereas the other group of mice was gavaged with B-DIM (5 mg/kg body weight, daily) for 4 wks. On termination, Matrigel plugs with tumor cells were harvested and fixed in 10% formalin, dehydrated, and embedded in paraffin. Matrigel plug slices were stained with H&E (left) and anti-CD31 antibody (middle and right). SPOT Advanced Imaging software was used to capture bright-field images. Histologic pictures of peripheral (middle) and deeper (right) areas of Matrigel plugs. Original magnification (×200). D, image analyses of the area of CD31-positive vascular, vascular number, and length of vascular perimeter in each of the entire field were carried out using software image analysis program. *, P < 0.05; **, P < 0.01 compared with control n = 4.
Volume 67, Issue 7
