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Urotensin II receptor determines prognosis of bladder cancer regulating cell motility/invasion.

Franco R, Zappavigna S, Gigantino V, Luce A, Cantile M, Cerrone M, Facchini G, Perdonà S, Pignata S, Di Lorenzo G, Chieffi S, Vitale G, De Sio M, Sgambato A, Botti G, Yousif AM, Novellino E, Grieco P, Caraglia M - J. Exp. Clin. Cancer Res. (2014)

Bottom Line: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC.Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells.High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy. michele.caraglia@unina2.it.

ABSTRACT

Background: Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC)/invasive have different gene profile and clinical course. NMIBC prognosis is not completely predictable, since the relapse rate is higher than 20%, even in the form of MIBC. The aim of this study is to evaluate if UTR expression can discriminate between NMIBC and MIBC and predict the risk of relapses in NMIBCs.

Methods: We have investigated upon urotensin-II (UII) receptor (UTR) expression in vivo in 159 patients affected by NMIBC. The biological role of UTR was also investigated in vitro. UTR expression was evaluated in a tissue-micro-array, consisting of normal, NMIBC and invasive bTCC samples.

Results: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC. The superagonist UPG84 induced growth suppression at nM concentrations on 3/4 cell lines. Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells.

Conclusions: The evaluation of UTR expression can discriminate between NMIBC at high and low risk of relapse. Moreover, our data suggest that UTR is involved in the regulation of motility, invasion and proliferation of bladder cancer cells. High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

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Effects of down-regulation or block of UTR with either an anti-UTR shRNA or the specific antagonist urantide on cell motility and invasion of RT112 cells. A: RT112 parental (CTR) or transiently transfected with a shRNA for UTR (shUTR), were plated in the top chamber of non-coated polyethylene teraphthalate (PET) membranes, treated or not with 100 nM urantide for 48 h and cell motility was evaluated as described in Methods Section. B: For in vitro invasion assays, RT112 cells were added to a Boyden chamber coated with Matrigel and cell invasion was evaluated as described in Methods Section. The migrating and the invading cells were stained with 0.25% crystal violet for 10 min and photographed under a microscope. C: The histogram shows the quantification of the migrating and invading cells measured with a spectrophotometer as OD, and the results are expressed as a percentage as compared to untreated RT112 parental cells. The experiments were performed three different times and the results are the mean of the obtained values. Bars, SDs. CTR, untreated RT112 cells; Sc, RT112 cells transfected with scrambled vector and cultured for 48 h; Sc + UR, RT112 cells transfected with scrambled vector and exposed for 48 h to 100 nM urantide; shUTR, RT112 cells transfected with shUTR and cultured for 48 h; shUTR + UR, RT112 cells transfected with shUTR and exposed to 100 nM urantide for 48 h. Asterisks indicate the statistical significance of the data (P <0.005).
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Figure 9: Effects of down-regulation or block of UTR with either an anti-UTR shRNA or the specific antagonist urantide on cell motility and invasion of RT112 cells. A: RT112 parental (CTR) or transiently transfected with a shRNA for UTR (shUTR), were plated in the top chamber of non-coated polyethylene teraphthalate (PET) membranes, treated or not with 100 nM urantide for 48 h and cell motility was evaluated as described in Methods Section. B: For in vitro invasion assays, RT112 cells were added to a Boyden chamber coated with Matrigel and cell invasion was evaluated as described in Methods Section. The migrating and the invading cells were stained with 0.25% crystal violet for 10 min and photographed under a microscope. C: The histogram shows the quantification of the migrating and invading cells measured with a spectrophotometer as OD, and the results are expressed as a percentage as compared to untreated RT112 parental cells. The experiments were performed three different times and the results are the mean of the obtained values. Bars, SDs. CTR, untreated RT112 cells; Sc, RT112 cells transfected with scrambled vector and cultured for 48 h; Sc + UR, RT112 cells transfected with scrambled vector and exposed for 48 h to 100 nM urantide; shUTR, RT112 cells transfected with shUTR and cultured for 48 h; shUTR + UR, RT112 cells transfected with shUTR and exposed to 100 nM urantide for 48 h. Asterisks indicate the statistical significance of the data (P <0.005).

Mentions: In order to explore the specific contribution of UTR in regulation of motility and invasion of bladder cancer cells, T24 and RT112 cells were treated with urantide (100 nM) for 48 h and/or were transiently transfected with shRNA for UTR to down-regulate UTR protein expression. Cells were seeded in transwell chambers and allowed to migrate and invade in absence or presence of urantide. After 48 h, urantide induced an about 35% and 50% reduction of cell motility and invasion, respectively, in T24 cells if compared to untreated cells. When T24 cells were transfected with shUTR displayed 45% and 56% inhibition of their ability to migrate and invade, respectively (Figure 8A and B, respectively). Downregulation of UTR in T24 treated with urantide did not increase the effect induced by urantide alone (Figure 8A and B, respectively). Similar results were also obtained in RT112 (Figure 9A and B). These data demonstrated that UTR is involved in both motility and invasion of human bladder cancer cells.


Urotensin II receptor determines prognosis of bladder cancer regulating cell motility/invasion.

Franco R, Zappavigna S, Gigantino V, Luce A, Cantile M, Cerrone M, Facchini G, Perdonà S, Pignata S, Di Lorenzo G, Chieffi S, Vitale G, De Sio M, Sgambato A, Botti G, Yousif AM, Novellino E, Grieco P, Caraglia M - J. Exp. Clin. Cancer Res. (2014)

Effects of down-regulation or block of UTR with either an anti-UTR shRNA or the specific antagonist urantide on cell motility and invasion of RT112 cells. A: RT112 parental (CTR) or transiently transfected with a shRNA for UTR (shUTR), were plated in the top chamber of non-coated polyethylene teraphthalate (PET) membranes, treated or not with 100 nM urantide for 48 h and cell motility was evaluated as described in Methods Section. B: For in vitro invasion assays, RT112 cells were added to a Boyden chamber coated with Matrigel and cell invasion was evaluated as described in Methods Section. The migrating and the invading cells were stained with 0.25% crystal violet for 10 min and photographed under a microscope. C: The histogram shows the quantification of the migrating and invading cells measured with a spectrophotometer as OD, and the results are expressed as a percentage as compared to untreated RT112 parental cells. The experiments were performed three different times and the results are the mean of the obtained values. Bars, SDs. CTR, untreated RT112 cells; Sc, RT112 cells transfected with scrambled vector and cultured for 48 h; Sc + UR, RT112 cells transfected with scrambled vector and exposed for 48 h to 100 nM urantide; shUTR, RT112 cells transfected with shUTR and cultured for 48 h; shUTR + UR, RT112 cells transfected with shUTR and exposed to 100 nM urantide for 48 h. Asterisks indicate the statistical significance of the data (P <0.005).
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Related In: Results  -  Collection

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Figure 9: Effects of down-regulation or block of UTR with either an anti-UTR shRNA or the specific antagonist urantide on cell motility and invasion of RT112 cells. A: RT112 parental (CTR) or transiently transfected with a shRNA for UTR (shUTR), were plated in the top chamber of non-coated polyethylene teraphthalate (PET) membranes, treated or not with 100 nM urantide for 48 h and cell motility was evaluated as described in Methods Section. B: For in vitro invasion assays, RT112 cells were added to a Boyden chamber coated with Matrigel and cell invasion was evaluated as described in Methods Section. The migrating and the invading cells were stained with 0.25% crystal violet for 10 min and photographed under a microscope. C: The histogram shows the quantification of the migrating and invading cells measured with a spectrophotometer as OD, and the results are expressed as a percentage as compared to untreated RT112 parental cells. The experiments were performed three different times and the results are the mean of the obtained values. Bars, SDs. CTR, untreated RT112 cells; Sc, RT112 cells transfected with scrambled vector and cultured for 48 h; Sc + UR, RT112 cells transfected with scrambled vector and exposed for 48 h to 100 nM urantide; shUTR, RT112 cells transfected with shUTR and cultured for 48 h; shUTR + UR, RT112 cells transfected with shUTR and exposed to 100 nM urantide for 48 h. Asterisks indicate the statistical significance of the data (P <0.005).
Mentions: In order to explore the specific contribution of UTR in regulation of motility and invasion of bladder cancer cells, T24 and RT112 cells were treated with urantide (100 nM) for 48 h and/or were transiently transfected with shRNA for UTR to down-regulate UTR protein expression. Cells were seeded in transwell chambers and allowed to migrate and invade in absence or presence of urantide. After 48 h, urantide induced an about 35% and 50% reduction of cell motility and invasion, respectively, in T24 cells if compared to untreated cells. When T24 cells were transfected with shUTR displayed 45% and 56% inhibition of their ability to migrate and invade, respectively (Figure 8A and B, respectively). Downregulation of UTR in T24 treated with urantide did not increase the effect induced by urantide alone (Figure 8A and B, respectively). Similar results were also obtained in RT112 (Figure 9A and B). These data demonstrated that UTR is involved in both motility and invasion of human bladder cancer cells.

Bottom Line: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC.Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells.High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, Naples, Italy. michele.caraglia@unina2.it.

ABSTRACT

Background: Non Muscle Invasive Bladder Transitional Cancer (NMIBC) and Muscle Invasive Bladder Transitional Cancer (MIBC)/invasive have different gene profile and clinical course. NMIBC prognosis is not completely predictable, since the relapse rate is higher than 20%, even in the form of MIBC. The aim of this study is to evaluate if UTR expression can discriminate between NMIBC and MIBC and predict the risk of relapses in NMIBCs.

Methods: We have investigated upon urotensin-II (UII) receptor (UTR) expression in vivo in 159 patients affected by NMIBC. The biological role of UTR was also investigated in vitro. UTR expression was evaluated in a tissue-micro-array, consisting of normal, NMIBC and invasive bTCC samples.

Results: UTR discriminated between NMIBC and MIBC and showed a significant correlation between low UTR expression and shorter disease free survival in NMIBC. The superagonist UPG84 induced growth suppression at nM concentrations on 3/4 cell lines. Bladder cancer cell treatment with the antagonist urantide or the knock-down of UTR with a specific shRNA significantly blocked both the motility and invasion of bladder cancer cells.

Conclusions: The evaluation of UTR expression can discriminate between NMIBC at high and low risk of relapse. Moreover, our data suggest that UTR is involved in the regulation of motility, invasion and proliferation of bladder cancer cells. High UTR expression is an independent prognostic factor of good prognosis for NMIBC regulating motility and invasion of bladder cancer cells.

Show MeSH
Related in: MedlinePlus