Limits...
Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination.

Kumar S, Kumar S, Rajendran M, Alam SM, Lin FF, Cheng PW, Lin MF - PLoS ONE (2011)

Bottom Line: These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist.Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT

Background: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.

Methods and findings: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.

Conclusions: The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

Show MeSH

Related in: MedlinePlus

Effects of steroids on p66Shc protein levels in PCa and OVa cells and their proliferation.LNCaP C-33 and CAOV-3 cells were plated in duplicates in the regular medium as described in the methods for 72 h and then maintained in a steroid-reduced medium for 48 h. Cells were treated with steroids plus or minus the corresponding antagonists as specified in each figure for 48 h. After harvested, lysates were analyzed by western blotting with Abs against total Shc and β-actin protein, respectively. The level of β-actin protein was detected as a loading control. The intensity of each Shc protein hybridization band was semiquantified, and the ratio to the corresponding β-actin protein was calculated and then normalized to that of corresponding control cells which received the solvent alone. To determine cell growth, cell number was analyzed by cell counting. The figure is a representative of three sets of independent experiments in duplicates and the cell growth is expressed as means ± SE (n = 3). (A) LNCaP C-33 cells were treated with or without DHT (10 nM), plus or minus casodex (10 µM) for 48 h. Left panel: Lane 1, cells received EtOH as a control; Lane 2, cells received DMSO as a control; Lane 3, cells received DHT in EtOH; Lane 4, cells received casodex in DMSO; Lane 5, cells received both DHT & casodex. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, **P<0.001). (B) Left panel: CaOV-3 cells treated without or with E2 (10 nM), tamoxifen (0.1 and 1 µM) for 48 h. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, *P<0.05). SD, Standard deviation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3021521&req=5

pone-0015942-g001: Effects of steroids on p66Shc protein levels in PCa and OVa cells and their proliferation.LNCaP C-33 and CAOV-3 cells were plated in duplicates in the regular medium as described in the methods for 72 h and then maintained in a steroid-reduced medium for 48 h. Cells were treated with steroids plus or minus the corresponding antagonists as specified in each figure for 48 h. After harvested, lysates were analyzed by western blotting with Abs against total Shc and β-actin protein, respectively. The level of β-actin protein was detected as a loading control. The intensity of each Shc protein hybridization band was semiquantified, and the ratio to the corresponding β-actin protein was calculated and then normalized to that of corresponding control cells which received the solvent alone. To determine cell growth, cell number was analyzed by cell counting. The figure is a representative of three sets of independent experiments in duplicates and the cell growth is expressed as means ± SE (n = 3). (A) LNCaP C-33 cells were treated with or without DHT (10 nM), plus or minus casodex (10 µM) for 48 h. Left panel: Lane 1, cells received EtOH as a control; Lane 2, cells received DMSO as a control; Lane 3, cells received DHT in EtOH; Lane 4, cells received casodex in DMSO; Lane 5, cells received both DHT & casodex. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, **P<0.001). (B) Left panel: CaOV-3 cells treated without or with E2 (10 nM), tamoxifen (0.1 and 1 µM) for 48 h. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, *P<0.05). SD, Standard deviation.

Mentions: LNCaP C-33 cells are androgen-sensitive cells with a slow growth rate and express a low level of p66Shc protein. Since p66Shc protein level can be up-regulated by steroid hormones [14], we investigated the molecular mechanism by which p66Shc protein is elevated in DHT-treated LNCaP cells. As shown in Fig. 1A (lane #3 of left panel), in DHT-treated LNCaP C-33 cells, p66Shc protein levels, but not p52 or p46 Shc protein, were elevated as observed previously [14]. The hybridization band was semiquantified by scanning and then normalizing to that of β-actin. The data validated the specific DHT effect on p66Shc protein, but not p52 or p46Shc, by over 2-fold elevation (lane #3, Fig. 1A). Concurrently, the proliferation of DHT-treated cells was increased (Fig. 1A, column #3 of right panel). On contrast, in the presence of casodex, an AR antagonist used in clinical androgen ablation therapy, the elevation of p66Shc protein by DHT was abolished (Fig. 1A, lane #5 of left panel) and cell growth was diminished (Fig. 1A, column #5 of right panel). Thus, casodex blocks the up-regulatory effect of DHT on p66Shc protein as well as the growth of LNCaP C-33 cells. The data indicate that AR activity is required for androgen-mediated up-regulation of p66Shc protein level.


Steroids up-regulate p66Shc longevity protein in growth regulation by inhibiting its ubiquitination.

Kumar S, Kumar S, Rajendran M, Alam SM, Lin FF, Cheng PW, Lin MF - PLoS ONE (2011)

Effects of steroids on p66Shc protein levels in PCa and OVa cells and their proliferation.LNCaP C-33 and CAOV-3 cells were plated in duplicates in the regular medium as described in the methods for 72 h and then maintained in a steroid-reduced medium for 48 h. Cells were treated with steroids plus or minus the corresponding antagonists as specified in each figure for 48 h. After harvested, lysates were analyzed by western blotting with Abs against total Shc and β-actin protein, respectively. The level of β-actin protein was detected as a loading control. The intensity of each Shc protein hybridization band was semiquantified, and the ratio to the corresponding β-actin protein was calculated and then normalized to that of corresponding control cells which received the solvent alone. To determine cell growth, cell number was analyzed by cell counting. The figure is a representative of three sets of independent experiments in duplicates and the cell growth is expressed as means ± SE (n = 3). (A) LNCaP C-33 cells were treated with or without DHT (10 nM), plus or minus casodex (10 µM) for 48 h. Left panel: Lane 1, cells received EtOH as a control; Lane 2, cells received DMSO as a control; Lane 3, cells received DHT in EtOH; Lane 4, cells received casodex in DMSO; Lane 5, cells received both DHT & casodex. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, **P<0.001). (B) Left panel: CaOV-3 cells treated without or with E2 (10 nM), tamoxifen (0.1 and 1 µM) for 48 h. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, *P<0.05). SD, Standard deviation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021521&req=5

pone-0015942-g001: Effects of steroids on p66Shc protein levels in PCa and OVa cells and their proliferation.LNCaP C-33 and CAOV-3 cells were plated in duplicates in the regular medium as described in the methods for 72 h and then maintained in a steroid-reduced medium for 48 h. Cells were treated with steroids plus or minus the corresponding antagonists as specified in each figure for 48 h. After harvested, lysates were analyzed by western blotting with Abs against total Shc and β-actin protein, respectively. The level of β-actin protein was detected as a loading control. The intensity of each Shc protein hybridization band was semiquantified, and the ratio to the corresponding β-actin protein was calculated and then normalized to that of corresponding control cells which received the solvent alone. To determine cell growth, cell number was analyzed by cell counting. The figure is a representative of three sets of independent experiments in duplicates and the cell growth is expressed as means ± SE (n = 3). (A) LNCaP C-33 cells were treated with or without DHT (10 nM), plus or minus casodex (10 µM) for 48 h. Left panel: Lane 1, cells received EtOH as a control; Lane 2, cells received DMSO as a control; Lane 3, cells received DHT in EtOH; Lane 4, cells received casodex in DMSO; Lane 5, cells received both DHT & casodex. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, **P<0.001). (B) Left panel: CaOV-3 cells treated without or with E2 (10 nM), tamoxifen (0.1 and 1 µM) for 48 h. Right panel: Cell growth was analyzed by cell counting. (n = 2×3, *P<0.05). SD, Standard deviation.
Mentions: LNCaP C-33 cells are androgen-sensitive cells with a slow growth rate and express a low level of p66Shc protein. Since p66Shc protein level can be up-regulated by steroid hormones [14], we investigated the molecular mechanism by which p66Shc protein is elevated in DHT-treated LNCaP cells. As shown in Fig. 1A (lane #3 of left panel), in DHT-treated LNCaP C-33 cells, p66Shc protein levels, but not p52 or p46 Shc protein, were elevated as observed previously [14]. The hybridization band was semiquantified by scanning and then normalizing to that of β-actin. The data validated the specific DHT effect on p66Shc protein, but not p52 or p46Shc, by over 2-fold elevation (lane #3, Fig. 1A). Concurrently, the proliferation of DHT-treated cells was increased (Fig. 1A, column #3 of right panel). On contrast, in the presence of casodex, an AR antagonist used in clinical androgen ablation therapy, the elevation of p66Shc protein by DHT was abolished (Fig. 1A, lane #5 of left panel) and cell growth was diminished (Fig. 1A, column #5 of right panel). Thus, casodex blocks the up-regulatory effect of DHT on p66Shc protein as well as the growth of LNCaP C-33 cells. The data indicate that AR activity is required for androgen-mediated up-regulation of p66Shc protein level.

Bottom Line: These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist.Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, College of Medicine, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

ABSTRACT

Background: p66Shc, an isoform of Shc adaptor proteins, mediates diverse signals, including cellular stress and mouse longevity. p66Shc protein level is elevated in several carcinomas and steroid-treated human cancer cells. Several lines of evidence indicate that p66Shc plays a critical role in steroid-related carcinogenesis, and steroids play a role in its elevated levels in those cells without known mechanism.

Methods and findings: In this study, we investigated the molecular mechanism by which steroid hormones up-regulate p66Shc protein level. In steroid-treated human prostate and ovarian cancer cells, p66Shc protein levels were elevated, correlating with increased cell proliferation. These steroid effects on p66Shc protein and cell growth were competed out by the respective antagonist. Further, actinomycin D and cyclohexamide could only partially block the elevated p66Shc protein level by steroids. Treatment with proteasomal inhibitors, but not lysosomal protease inhibitor, resulted in elevated p66Shc protein levels, even higher than that by steroids. Using prostate cancer cells as a model, immunoprecipitation revealed that androgens and proteasomal inhibitors reduce the ubiquitinated p66Shc proteins.

Conclusions: The data collectively indicate that functional steroid receptors are required in steroid up-regulation of p66Shc protein levels in prostate and ovarian cancer cells, correlating with cell proliferation. In these steroid-treated cells, elevated p66Shc protein level is apparently in part due to inhibiting its ubiquitination. The results may lead to an impact on advanced cancer therapy via the regulation of p66Shc protein by up-regulating its ubiquitination pathway.

Show MeSH
Related in: MedlinePlus