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Regulation of bombesin-stimulated cyclooxygenase-2 expression in prostate cancer cells.

Wen X, Chao C, Ives K, Hellmich MR - BMC Mol. Biol. (2011)

Bottom Line: Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression.The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA.Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways.

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Affiliation: Department of Surgery, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555, USA.

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The PI3K/Akt pathway regulates BBS-stimulated COX-2 promoter activity and AP-1 activation. PC-3 cells were co-transfected with plasmids containing the 1.4 kb human COX-2 promoter 5' of a luciferase reporter gene and β-galactosidase. A) Twenty-four hours after transfection, cells were treated either with vehicle or 10 nM BBS for 2, 4, and 6 h. Changes in luciferase activity (mean ± S.D, n = 3) are expressed relative to vehicle treated at each time point after normalizing for transfection efficiency using β-galactosidase activity [* BBS vs. time-point matched vehicle control, p < 0.05]. B) Pre-treatment with LY294002 (25 μM) partially inhibited BBS-stimulated COX-2 promoter activity [* LY294002 vs. BBS alone, p < 0.05, n = 3], whereas SB203580 (10 μM) had no effect. C) Autoradiogram showing binding of nuclear proteins to 32P-labeled oligonucleotide containing the AP-1 consensus sequence [lane 1, radiolabeled probe only; lane 2, nuclear proteins + radio-labeled probe and excess unlabeled probe; lane 3, nuclear protein from vehicle-treated cells; lane 4 nuclear proteins from cells treated with BBS (10 nM) for 30 min; lanes 5 and 6, nuclear proteins from cells pretreated with LY294002 (25 μM) for 30 min followed by vehicle or BBS for 30 min]. D) PC-3 cells were treated with BBS (10 nM) or TNF-α (10 ng/ml) alone or in combination with inhibitors for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Quantification of the effects of curcumin (Cur) (20 μM) and LY294002 (LY) (25 μM) on BBS-stimulated NF-κB nuclear translocation. Data are expressed as the ratio of NF-κB-positive stained nuclei divided by the total number of cells per high power field (200X). Four fields were counted for each condition from 3 independent experiment [† BBS or BBS + LY294002 vs. vehicle or LY294002 alone, p ≤ 0.001; * BBS + curcumin vs. BBS alone or TNFα + curcumin vs. TNFα alone, p ≤ 0.001].
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Figure 4: The PI3K/Akt pathway regulates BBS-stimulated COX-2 promoter activity and AP-1 activation. PC-3 cells were co-transfected with plasmids containing the 1.4 kb human COX-2 promoter 5' of a luciferase reporter gene and β-galactosidase. A) Twenty-four hours after transfection, cells were treated either with vehicle or 10 nM BBS for 2, 4, and 6 h. Changes in luciferase activity (mean ± S.D, n = 3) are expressed relative to vehicle treated at each time point after normalizing for transfection efficiency using β-galactosidase activity [* BBS vs. time-point matched vehicle control, p < 0.05]. B) Pre-treatment with LY294002 (25 μM) partially inhibited BBS-stimulated COX-2 promoter activity [* LY294002 vs. BBS alone, p < 0.05, n = 3], whereas SB203580 (10 μM) had no effect. C) Autoradiogram showing binding of nuclear proteins to 32P-labeled oligonucleotide containing the AP-1 consensus sequence [lane 1, radiolabeled probe only; lane 2, nuclear proteins + radio-labeled probe and excess unlabeled probe; lane 3, nuclear protein from vehicle-treated cells; lane 4 nuclear proteins from cells treated with BBS (10 nM) for 30 min; lanes 5 and 6, nuclear proteins from cells pretreated with LY294002 (25 μM) for 30 min followed by vehicle or BBS for 30 min]. D) PC-3 cells were treated with BBS (10 nM) or TNF-α (10 ng/ml) alone or in combination with inhibitors for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Quantification of the effects of curcumin (Cur) (20 μM) and LY294002 (LY) (25 μM) on BBS-stimulated NF-κB nuclear translocation. Data are expressed as the ratio of NF-κB-positive stained nuclei divided by the total number of cells per high power field (200X). Four fields were counted for each condition from 3 independent experiment [† BBS or BBS + LY294002 vs. vehicle or LY294002 alone, p ≤ 0.001; * BBS + curcumin vs. BBS alone or TNFα + curcumin vs. TNFα alone, p ≤ 0.001].

Mentions: The cellular levels of COX-2 mRNA can be regulated both by enhanced gene transcription and inhibition of message degradation [31,32]. To determine whether BBS treatment enhanced COX-2 gene transcription, PC-3 cells were first transiently transfected with a transcription reporter construct consisting of 1.4 kb of the human COX-2 promoter coupled to a luciferase gene and then stimulated with BBS over a time course. BBS induced a time-dependent increase (1.4- to 2.3-fold) in COX-2 promoter activity when compared to vehicle-treated control cell cultures (Figure 4A). To determine whether the p38MAPK or PI3K/Akt pathways were involved in BBS-stimulated COX-2 transcription, cells were pretreated with SB203580 (10 μM) or LY294002 (25 μM) for 30 min followed by a 6-h treatment with BBS (10 nM). Compared to BBS treatment alone, LY294002 inhibited approximately 50% of the increase in BBS-stimulated luciferase activity (Figure 4B). In contrast, SB203580 had no effect on BBS-stimulated COX-2 promoter activity (Figure 4B), suggesting that the PI3K/Akt pathway, not the p38MAPK pathway, is involved in BBS-induced COX-2 gene transcription in PC-3 cells.


Regulation of bombesin-stimulated cyclooxygenase-2 expression in prostate cancer cells.

Wen X, Chao C, Ives K, Hellmich MR - BMC Mol. Biol. (2011)

The PI3K/Akt pathway regulates BBS-stimulated COX-2 promoter activity and AP-1 activation. PC-3 cells were co-transfected with plasmids containing the 1.4 kb human COX-2 promoter 5' of a luciferase reporter gene and β-galactosidase. A) Twenty-four hours after transfection, cells were treated either with vehicle or 10 nM BBS for 2, 4, and 6 h. Changes in luciferase activity (mean ± S.D, n = 3) are expressed relative to vehicle treated at each time point after normalizing for transfection efficiency using β-galactosidase activity [* BBS vs. time-point matched vehicle control, p < 0.05]. B) Pre-treatment with LY294002 (25 μM) partially inhibited BBS-stimulated COX-2 promoter activity [* LY294002 vs. BBS alone, p < 0.05, n = 3], whereas SB203580 (10 μM) had no effect. C) Autoradiogram showing binding of nuclear proteins to 32P-labeled oligonucleotide containing the AP-1 consensus sequence [lane 1, radiolabeled probe only; lane 2, nuclear proteins + radio-labeled probe and excess unlabeled probe; lane 3, nuclear protein from vehicle-treated cells; lane 4 nuclear proteins from cells treated with BBS (10 nM) for 30 min; lanes 5 and 6, nuclear proteins from cells pretreated with LY294002 (25 μM) for 30 min followed by vehicle or BBS for 30 min]. D) PC-3 cells were treated with BBS (10 nM) or TNF-α (10 ng/ml) alone or in combination with inhibitors for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Quantification of the effects of curcumin (Cur) (20 μM) and LY294002 (LY) (25 μM) on BBS-stimulated NF-κB nuclear translocation. Data are expressed as the ratio of NF-κB-positive stained nuclei divided by the total number of cells per high power field (200X). Four fields were counted for each condition from 3 independent experiment [† BBS or BBS + LY294002 vs. vehicle or LY294002 alone, p ≤ 0.001; * BBS + curcumin vs. BBS alone or TNFα + curcumin vs. TNFα alone, p ≤ 0.001].
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Figure 4: The PI3K/Akt pathway regulates BBS-stimulated COX-2 promoter activity and AP-1 activation. PC-3 cells were co-transfected with plasmids containing the 1.4 kb human COX-2 promoter 5' of a luciferase reporter gene and β-galactosidase. A) Twenty-four hours after transfection, cells were treated either with vehicle or 10 nM BBS for 2, 4, and 6 h. Changes in luciferase activity (mean ± S.D, n = 3) are expressed relative to vehicle treated at each time point after normalizing for transfection efficiency using β-galactosidase activity [* BBS vs. time-point matched vehicle control, p < 0.05]. B) Pre-treatment with LY294002 (25 μM) partially inhibited BBS-stimulated COX-2 promoter activity [* LY294002 vs. BBS alone, p < 0.05, n = 3], whereas SB203580 (10 μM) had no effect. C) Autoradiogram showing binding of nuclear proteins to 32P-labeled oligonucleotide containing the AP-1 consensus sequence [lane 1, radiolabeled probe only; lane 2, nuclear proteins + radio-labeled probe and excess unlabeled probe; lane 3, nuclear protein from vehicle-treated cells; lane 4 nuclear proteins from cells treated with BBS (10 nM) for 30 min; lanes 5 and 6, nuclear proteins from cells pretreated with LY294002 (25 μM) for 30 min followed by vehicle or BBS for 30 min]. D) PC-3 cells were treated with BBS (10 nM) or TNF-α (10 ng/ml) alone or in combination with inhibitors for 30 min, fixed, and immunostained with an antibody to the p65 subunit of NF-κB. Quantification of the effects of curcumin (Cur) (20 μM) and LY294002 (LY) (25 μM) on BBS-stimulated NF-κB nuclear translocation. Data are expressed as the ratio of NF-κB-positive stained nuclei divided by the total number of cells per high power field (200X). Four fields were counted for each condition from 3 independent experiment [† BBS or BBS + LY294002 vs. vehicle or LY294002 alone, p ≤ 0.001; * BBS + curcumin vs. BBS alone or TNFα + curcumin vs. TNFα alone, p ≤ 0.001].
Mentions: The cellular levels of COX-2 mRNA can be regulated both by enhanced gene transcription and inhibition of message degradation [31,32]. To determine whether BBS treatment enhanced COX-2 gene transcription, PC-3 cells were first transiently transfected with a transcription reporter construct consisting of 1.4 kb of the human COX-2 promoter coupled to a luciferase gene and then stimulated with BBS over a time course. BBS induced a time-dependent increase (1.4- to 2.3-fold) in COX-2 promoter activity when compared to vehicle-treated control cell cultures (Figure 4A). To determine whether the p38MAPK or PI3K/Akt pathways were involved in BBS-stimulated COX-2 transcription, cells were pretreated with SB203580 (10 μM) or LY294002 (25 μM) for 30 min followed by a 6-h treatment with BBS (10 nM). Compared to BBS treatment alone, LY294002 inhibited approximately 50% of the increase in BBS-stimulated luciferase activity (Figure 4B). In contrast, SB203580 had no effect on BBS-stimulated COX-2 promoter activity (Figure 4B), suggesting that the PI3K/Akt pathway, not the p38MAPK pathway, is involved in BBS-induced COX-2 gene transcription in PC-3 cells.

Bottom Line: Although BBS stimulates nuclear factor-kappaB (NF-κB) in PC-3, NF-κB does not regulate GRPR-mediated COX-2 expression.The p38MAPK pathway increases BBS-stimulated COX-2 expression by slowing the degradation of COX-2 mRNA.Expression of recombinant GRPR in the androgen-sensitive cell line LNCaP is sufficient to confer BBS-stimulated COX-2 expression via the p38MAPK and PI3K/Akt pathways.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, Univ. of Texas Medical Branch, 301 Univ. Blvd., Galveston, TX 77555, USA.

Show MeSH
Related in: MedlinePlus