Limits...
Methylseleninic acid elevates REDD1 and inhibits prostate cancer cell growth despite AKT activation and mTOR dysregulation in hypoxia.

Sinha I, Allen JE, Pinto JT, Sinha R - Cancer Med (2014)

Bottom Line: We have now extended these studies to evaluate the impact of MSeA on REDD1 (an mTOR inhibitor) in inducing cell death of invasive prostate cancer cells in hypoxia.Furthermore, REDD1 induction by MSeA is independent of AKT and the mTOR inhibition in prostate cancer cells causes partial resistance to MSeA-induced growth reduction in hypoxia.Our data suggest that MSeA induces REDD1 and inhibits prostate cancer cell growth in hypoxia despite activation of AKT and dysregulation of mTOR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Penn State Hershey Cancer Institute, Hershey, Pennsylvania.

Show MeSH

Related in: MedlinePlus

Protein expression changes in cytosolic fractions of PC3, DU145, and PC-3M cells following MSeA treatments for 2 h, 6 h and overnight in hypoxia. The three prostate cancer cell types were stimulated with 10% FBS following an overnight starvation (0.1% FBS) in the presence and absence of MSeA in hypoxia (1% O2). (A) Relatively increased levels of pAKT, REDD1 and pp70S6K (lower band) were observed after 2 h of MSeA treatment in invasive prostate cancer cells in hypoxia compared to respective untreated controls. (B) MSeA treatment after 6 h maintains elevated levels of pAKT in PC3 and PC-3M cells. REDD1 and pp70S6K levels induced by MSeA continue to increase in the three cell types at this time point. (C) An overnight exposure of invasive prostate cancer cells to MSeA maintained high levels of pAKT in PC3 and PC-3M cells while pAKT protein expression was marginally detectable in DU145 cells. The increase in REDD1 levels are more pronounced in the DU145 and PC-3M cells at this time point and pp70S6K expression was relatively higher in MSeA-treated cells as compared to untreated controls in each cell line. In addition, cleaved PARP appeared in a dose-dependent manner in each of the invasive prostate cancer cells following an overnight exposure with MSeA in hypoxia. (D) Following a 6 h treatment with MSeA in PC-3M cells, a concomitant dose-dependent elevation in pGSK3β expression was observed. Fold change in band densities of phosphorylated proteins were normalized to the band densities of their respective native protein and to β-actin levels. While for pp70S6K, pGSK3β and REDD1 the band densities were normalized to β-actin levels. FBS, fetal bovine serum; MSeA, methylseleninic acid; PARP, poly ADP-ribose polymerase; REDD1, regulated in development and DNA damage 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3987075&req=5

fig01: Protein expression changes in cytosolic fractions of PC3, DU145, and PC-3M cells following MSeA treatments for 2 h, 6 h and overnight in hypoxia. The three prostate cancer cell types were stimulated with 10% FBS following an overnight starvation (0.1% FBS) in the presence and absence of MSeA in hypoxia (1% O2). (A) Relatively increased levels of pAKT, REDD1 and pp70S6K (lower band) were observed after 2 h of MSeA treatment in invasive prostate cancer cells in hypoxia compared to respective untreated controls. (B) MSeA treatment after 6 h maintains elevated levels of pAKT in PC3 and PC-3M cells. REDD1 and pp70S6K levels induced by MSeA continue to increase in the three cell types at this time point. (C) An overnight exposure of invasive prostate cancer cells to MSeA maintained high levels of pAKT in PC3 and PC-3M cells while pAKT protein expression was marginally detectable in DU145 cells. The increase in REDD1 levels are more pronounced in the DU145 and PC-3M cells at this time point and pp70S6K expression was relatively higher in MSeA-treated cells as compared to untreated controls in each cell line. In addition, cleaved PARP appeared in a dose-dependent manner in each of the invasive prostate cancer cells following an overnight exposure with MSeA in hypoxia. (D) Following a 6 h treatment with MSeA in PC-3M cells, a concomitant dose-dependent elevation in pGSK3β expression was observed. Fold change in band densities of phosphorylated proteins were normalized to the band densities of their respective native protein and to β-actin levels. While for pp70S6K, pGSK3β and REDD1 the band densities were normalized to β-actin levels. FBS, fetal bovine serum; MSeA, methylseleninic acid; PARP, poly ADP-ribose polymerase; REDD1, regulated in development and DNA damage 1.

Mentions: REDD1 regulates mTORC1 activity and the latter is critical for progression of prostate cancer disease. However, mTORC1 regulation by REDD1 has not been well studied in prostate cancer. We first investigated how MSeA impacts REDD1, AKT, and p70S6K (downstream target of mTORC1) in three different prostate cancer cell lines, DU145 (PTEN+), PC3, and PC-3M (PTEN−) at 2, 6 h and overnight (18 h) treatments with MSeA in hypoxia. Additionally, we used LNCaP cells but these did not survive in hypoxia and therefore were not included in further experiments. In DU-145 and PC-3M cells, MSeA upregulates REDD1 protein expression at all time points (Fig. 1A–C). However, in PC3 cells, REDD1 was increased by MSeA at 2 and 6 h time points but was stabilized by overnight treatment. In addition, pAKT levels are elevated following 2 h MSeA treatment in all the three cell types (Fig. 1A) and decrease only in DU145 cells at 6 h and overnight MSeA treatment (Fig. 1B and C). However, pAKT levels in control PC3 cells are increasing whereas in PC-3M control cells the pAKT levels are decreasing as a function of time in hypoxia. Similar observation has been reported by other investigators 41. Furthermore, MSeA-induced AKT activation in PC-3M cells was confirmed by phosphorylation of GSK3β in a dose-dependent manner after 6 h treatment (Fig. 1D). The phosphorylated p70S6K expression was elevated in all the prostate cancer cell lines at all time points following treatments with MSeA in hypoxia (Fig. 1A–C). The above events may play a role in MSeA-induced apoptosis (as measured by cleaved-PARP) in a dose-dependent manner in PC3, DU145, and PC-3M cells following overnight treatment (Fig. 1C).


Methylseleninic acid elevates REDD1 and inhibits prostate cancer cell growth despite AKT activation and mTOR dysregulation in hypoxia.

Sinha I, Allen JE, Pinto JT, Sinha R - Cancer Med (2014)

Protein expression changes in cytosolic fractions of PC3, DU145, and PC-3M cells following MSeA treatments for 2 h, 6 h and overnight in hypoxia. The three prostate cancer cell types were stimulated with 10% FBS following an overnight starvation (0.1% FBS) in the presence and absence of MSeA in hypoxia (1% O2). (A) Relatively increased levels of pAKT, REDD1 and pp70S6K (lower band) were observed after 2 h of MSeA treatment in invasive prostate cancer cells in hypoxia compared to respective untreated controls. (B) MSeA treatment after 6 h maintains elevated levels of pAKT in PC3 and PC-3M cells. REDD1 and pp70S6K levels induced by MSeA continue to increase in the three cell types at this time point. (C) An overnight exposure of invasive prostate cancer cells to MSeA maintained high levels of pAKT in PC3 and PC-3M cells while pAKT protein expression was marginally detectable in DU145 cells. The increase in REDD1 levels are more pronounced in the DU145 and PC-3M cells at this time point and pp70S6K expression was relatively higher in MSeA-treated cells as compared to untreated controls in each cell line. In addition, cleaved PARP appeared in a dose-dependent manner in each of the invasive prostate cancer cells following an overnight exposure with MSeA in hypoxia. (D) Following a 6 h treatment with MSeA in PC-3M cells, a concomitant dose-dependent elevation in pGSK3β expression was observed. Fold change in band densities of phosphorylated proteins were normalized to the band densities of their respective native protein and to β-actin levels. While for pp70S6K, pGSK3β and REDD1 the band densities were normalized to β-actin levels. FBS, fetal bovine serum; MSeA, methylseleninic acid; PARP, poly ADP-ribose polymerase; REDD1, regulated in development and DNA damage 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3987075&req=5

fig01: Protein expression changes in cytosolic fractions of PC3, DU145, and PC-3M cells following MSeA treatments for 2 h, 6 h and overnight in hypoxia. The three prostate cancer cell types were stimulated with 10% FBS following an overnight starvation (0.1% FBS) in the presence and absence of MSeA in hypoxia (1% O2). (A) Relatively increased levels of pAKT, REDD1 and pp70S6K (lower band) were observed after 2 h of MSeA treatment in invasive prostate cancer cells in hypoxia compared to respective untreated controls. (B) MSeA treatment after 6 h maintains elevated levels of pAKT in PC3 and PC-3M cells. REDD1 and pp70S6K levels induced by MSeA continue to increase in the three cell types at this time point. (C) An overnight exposure of invasive prostate cancer cells to MSeA maintained high levels of pAKT in PC3 and PC-3M cells while pAKT protein expression was marginally detectable in DU145 cells. The increase in REDD1 levels are more pronounced in the DU145 and PC-3M cells at this time point and pp70S6K expression was relatively higher in MSeA-treated cells as compared to untreated controls in each cell line. In addition, cleaved PARP appeared in a dose-dependent manner in each of the invasive prostate cancer cells following an overnight exposure with MSeA in hypoxia. (D) Following a 6 h treatment with MSeA in PC-3M cells, a concomitant dose-dependent elevation in pGSK3β expression was observed. Fold change in band densities of phosphorylated proteins were normalized to the band densities of their respective native protein and to β-actin levels. While for pp70S6K, pGSK3β and REDD1 the band densities were normalized to β-actin levels. FBS, fetal bovine serum; MSeA, methylseleninic acid; PARP, poly ADP-ribose polymerase; REDD1, regulated in development and DNA damage 1.
Mentions: REDD1 regulates mTORC1 activity and the latter is critical for progression of prostate cancer disease. However, mTORC1 regulation by REDD1 has not been well studied in prostate cancer. We first investigated how MSeA impacts REDD1, AKT, and p70S6K (downstream target of mTORC1) in three different prostate cancer cell lines, DU145 (PTEN+), PC3, and PC-3M (PTEN−) at 2, 6 h and overnight (18 h) treatments with MSeA in hypoxia. Additionally, we used LNCaP cells but these did not survive in hypoxia and therefore were not included in further experiments. In DU-145 and PC-3M cells, MSeA upregulates REDD1 protein expression at all time points (Fig. 1A–C). However, in PC3 cells, REDD1 was increased by MSeA at 2 and 6 h time points but was stabilized by overnight treatment. In addition, pAKT levels are elevated following 2 h MSeA treatment in all the three cell types (Fig. 1A) and decrease only in DU145 cells at 6 h and overnight MSeA treatment (Fig. 1B and C). However, pAKT levels in control PC3 cells are increasing whereas in PC-3M control cells the pAKT levels are decreasing as a function of time in hypoxia. Similar observation has been reported by other investigators 41. Furthermore, MSeA-induced AKT activation in PC-3M cells was confirmed by phosphorylation of GSK3β in a dose-dependent manner after 6 h treatment (Fig. 1D). The phosphorylated p70S6K expression was elevated in all the prostate cancer cell lines at all time points following treatments with MSeA in hypoxia (Fig. 1A–C). The above events may play a role in MSeA-induced apoptosis (as measured by cleaved-PARP) in a dose-dependent manner in PC3, DU145, and PC-3M cells following overnight treatment (Fig. 1C).

Bottom Line: We have now extended these studies to evaluate the impact of MSeA on REDD1 (an mTOR inhibitor) in inducing cell death of invasive prostate cancer cells in hypoxia.Furthermore, REDD1 induction by MSeA is independent of AKT and the mTOR inhibition in prostate cancer cells causes partial resistance to MSeA-induced growth reduction in hypoxia.Our data suggest that MSeA induces REDD1 and inhibits prostate cancer cell growth in hypoxia despite activation of AKT and dysregulation of mTOR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Penn State College of Medicine, Penn State Hershey Cancer Institute, Hershey, Pennsylvania.

Show MeSH
Related in: MedlinePlus