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Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB, Koul HK - Oncogene (2009)

Bottom Line: Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells.Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity.We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology, Department of Surgery, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.

ABSTRACT
Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia-reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

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Hypoxia stabilizes HIF-1α and VEGF in LNCaP cells(a) LNCaP cells were subjected to hypoxia in a modular incubator for 4h and 12h. Western blot analysis for HIF-1α was done using GAPDH protein as a loading control. (b) LNCaP cells were transfected with pGL3-TK-3X HRE plasmid and pRL-CMV as a transfection control. 48h post transfection cells were incubated in hypoxia for 12h and 24h. Luciferase activity was measured with Dual luciferase assay kit and the firefly luciferase activity was normalized to Renilla luciferase activity. Each data point represents mean ± S.D of duplicate experiments done in duplicate. (c) LNCaP cells were subjected to hypoxia for 4h and 6h and total RNA was isolated. Reverse Transcriptase PCR was performed to determine the mRNA levels of VEGF; GAPDH was used as a loading control.
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Figure 1: Hypoxia stabilizes HIF-1α and VEGF in LNCaP cells(a) LNCaP cells were subjected to hypoxia in a modular incubator for 4h and 12h. Western blot analysis for HIF-1α was done using GAPDH protein as a loading control. (b) LNCaP cells were transfected with pGL3-TK-3X HRE plasmid and pRL-CMV as a transfection control. 48h post transfection cells were incubated in hypoxia for 12h and 24h. Luciferase activity was measured with Dual luciferase assay kit and the firefly luciferase activity was normalized to Renilla luciferase activity. Each data point represents mean ± S.D of duplicate experiments done in duplicate. (c) LNCaP cells were subjected to hypoxia for 4h and 6h and total RNA was isolated. Reverse Transcriptase PCR was performed to determine the mRNA levels of VEGF; GAPDH was used as a loading control.

Mentions: It is well known that the primary response of cells to lower levels of oxygen is the stabilization and increased activity of Hypoxia Inducible Factor (HIF-1α). Since hypoxia response is mediated by HIF-1α, we investigated the changes in HIF-1α and its downstream target Vascular Endothelial Growth Factor (VEGF) upon exposing LNCaP cells to hypoxia. In as less as 4h in hypoxia, the amount of HIF-1α protein was increased, while it was undetectable in normoxic conditions (Figure 1a) in LNCaP cells. We used the Hypoxia Response Element (HRE) driven luciferase reporter (3X HRE-luc) to measure HIF-1α activity in LNCaP cells and observed a significant increase in the luciferase activity (Figure 1b) which indicated higher levels of active HIF-1α in LNCaP cells under hypoxia. As expected, higher luciferase activity was observed in LNCaP cells exposed to hypoxia for 24h than for 12h.


Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1alpha levels contribute to emergence of an aggressive phenotype in prostate cancer.

Khandrika L, Lieberman R, Koul S, Kumar B, Maroni P, Chandhoke R, Meacham RB, Koul HK - Oncogene (2009)

Hypoxia stabilizes HIF-1α and VEGF in LNCaP cells(a) LNCaP cells were subjected to hypoxia in a modular incubator for 4h and 12h. Western blot analysis for HIF-1α was done using GAPDH protein as a loading control. (b) LNCaP cells were transfected with pGL3-TK-3X HRE plasmid and pRL-CMV as a transfection control. 48h post transfection cells were incubated in hypoxia for 12h and 24h. Luciferase activity was measured with Dual luciferase assay kit and the firefly luciferase activity was normalized to Renilla luciferase activity. Each data point represents mean ± S.D of duplicate experiments done in duplicate. (c) LNCaP cells were subjected to hypoxia for 4h and 6h and total RNA was isolated. Reverse Transcriptase PCR was performed to determine the mRNA levels of VEGF; GAPDH was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2651999&req=5

Figure 1: Hypoxia stabilizes HIF-1α and VEGF in LNCaP cells(a) LNCaP cells were subjected to hypoxia in a modular incubator for 4h and 12h. Western blot analysis for HIF-1α was done using GAPDH protein as a loading control. (b) LNCaP cells were transfected with pGL3-TK-3X HRE plasmid and pRL-CMV as a transfection control. 48h post transfection cells were incubated in hypoxia for 12h and 24h. Luciferase activity was measured with Dual luciferase assay kit and the firefly luciferase activity was normalized to Renilla luciferase activity. Each data point represents mean ± S.D of duplicate experiments done in duplicate. (c) LNCaP cells were subjected to hypoxia for 4h and 6h and total RNA was isolated. Reverse Transcriptase PCR was performed to determine the mRNA levels of VEGF; GAPDH was used as a loading control.
Mentions: It is well known that the primary response of cells to lower levels of oxygen is the stabilization and increased activity of Hypoxia Inducible Factor (HIF-1α). Since hypoxia response is mediated by HIF-1α, we investigated the changes in HIF-1α and its downstream target Vascular Endothelial Growth Factor (VEGF) upon exposing LNCaP cells to hypoxia. In as less as 4h in hypoxia, the amount of HIF-1α protein was increased, while it was undetectable in normoxic conditions (Figure 1a) in LNCaP cells. We used the Hypoxia Response Element (HRE) driven luciferase reporter (3X HRE-luc) to measure HIF-1α activity in LNCaP cells and observed a significant increase in the luciferase activity (Figure 1b) which indicated higher levels of active HIF-1α in LNCaP cells under hypoxia. As expected, higher luciferase activity was observed in LNCaP cells exposed to hypoxia for 24h than for 12h.

Bottom Line: Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells.Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity.We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction and Molecular Urology Laboratory-Program in Urosciences, Division of Urology, Department of Surgery, School of Medicine, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.

ABSTRACT
Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia-reoxygenation, however, the role of hypoxia-reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia-reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia-reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia-reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1alpha) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia-reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia-reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

Show MeSH
Related in: MedlinePlus