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Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1α protein under hypoxic conditions.

Laemmle A, Lechleiter A, Roh V, Schwarz C, Portmann S, Furer C, Keogh A, Tschan MP, Candinas D, Vorburger SA, Stroka D - PLoS ONE (2012)

Bottom Line: Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2.Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes.In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Visceral Surgery and Medicine, Visceral and Transplantation Surgery, University Hospital Bern and University of Bern, Bern, Switzerland.

ABSTRACT
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

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Inhibition of SIRT1 with cambinol impairs hypoxic response in vivo.A. HepG2 cells were treated with 50 and 100 µM sirtinol or cambinol or an equivalent concentration of DMSO (0) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. A representative blot of 2 independently performed experiments is shown. B. Wild type C57BL/6 mice were administered 100 mg/kg cambinol 2 hours prior to exposure to 6% O2 for 6 hours. Total RNA was isolated from liver, kidney and brain tissues. RT-qPCR was performed and ΔCt values of EPO mRNA of hypoxic mice treated with vehicle or cambinol were compared the average delta Ct values of vehicle-treated normoxic mice. The fold change of EPO mRNA was from 221±65 (control) to 88±48 (cambinol), **p = 0.0014 in the kidney and from 120±43 (control) to 21±10.6 (cambinol), *p = 0.01 in the liver and from 9.6±2.7 (control) to 8.3±2.3 (cambinol), p = 0.5036 in the brain. Columns, mean values from 4 independent experiments ±SD. C. Relative luciferase units (RLU) were measured as an indicator of tumor size in mice harboring intrahepatic luciferase-labeled HepG2 tumors. Mice treated with 100 mg/kg cambinol had an overall smaller tumor volume compared to vehicle treated controls. Dots are representative of individual animals, bars are the mean ±SD. D. Total RNA was isolated from HepG2 tumors. ΔCt values of VEGF mRNA in vehicle treated mice were compared to mice treated with cambinol by RT-qPCR. Dots are representative of individual animals and bars are the mean ±SD. E. Haematoxylin and eosin stain of excised tumors from mice treated with vehicle or cambinol.
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pone-0033433-g005: Inhibition of SIRT1 with cambinol impairs hypoxic response in vivo.A. HepG2 cells were treated with 50 and 100 µM sirtinol or cambinol or an equivalent concentration of DMSO (0) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. A representative blot of 2 independently performed experiments is shown. B. Wild type C57BL/6 mice were administered 100 mg/kg cambinol 2 hours prior to exposure to 6% O2 for 6 hours. Total RNA was isolated from liver, kidney and brain tissues. RT-qPCR was performed and ΔCt values of EPO mRNA of hypoxic mice treated with vehicle or cambinol were compared the average delta Ct values of vehicle-treated normoxic mice. The fold change of EPO mRNA was from 221±65 (control) to 88±48 (cambinol), **p = 0.0014 in the kidney and from 120±43 (control) to 21±10.6 (cambinol), *p = 0.01 in the liver and from 9.6±2.7 (control) to 8.3±2.3 (cambinol), p = 0.5036 in the brain. Columns, mean values from 4 independent experiments ±SD. C. Relative luciferase units (RLU) were measured as an indicator of tumor size in mice harboring intrahepatic luciferase-labeled HepG2 tumors. Mice treated with 100 mg/kg cambinol had an overall smaller tumor volume compared to vehicle treated controls. Dots are representative of individual animals, bars are the mean ±SD. D. Total RNA was isolated from HepG2 tumors. ΔCt values of VEGF mRNA in vehicle treated mice were compared to mice treated with cambinol by RT-qPCR. Dots are representative of individual animals and bars are the mean ±SD. E. Haematoxylin and eosin stain of excised tumors from mice treated with vehicle or cambinol.

Mentions: We next tested if pharmacologically targeting SIRT1 can also influence HIF activity in vivo. Cambinol, a cell permeable β-naphthol compound inhibits the NAD+-dependent deacetylase activity of SIRT1 and SIRT2 (IC50 = 56 µM and 59 µM, respectively) and exhibits no inhibition against class I or II histone deacetylase activity [47]. Unlike sirtinol, cambinol can be used in vivo and was shown to effectively inhibit xenograft BCL6-expressing Burkitt lymphoma growth in mice [47]. In a first step, the suppressive effect of cambinol was tested and compared to sirtinol in HepG2 cells in vitro (Figure 5A). HepG2 cells are tumorigenic in immune deficient mice, therefore provide the opportunity to test the effect of SIRT1 inhibition in an HCC xenograft model. Inhibition of SIRT activity with cambinol led to a dose-dependent repression of HIF-1α protein accumulation in HepG2 cells in vitro. Cells treated with sirtinol were used for comparison (Figure 5A). We previously reported that in mice exposed to 6% oxygen HIF-1α protein accumulates in various tissues and activates HIF target genes [48]. Therefore, we tested if inhibition of SIRT1 represses a HIF-driven response in vivo. Mice were pre-treated with cambinol for 2 hours and then exposed to 6% oxygen for 6 hours. Analysis of mouse tissues showed that there was a significant decrease of EPO mRNA in the kidney and the liver in mice pre-treated with cambinol, whereas, pre-treatment with cambinol did not reduce EPO mRNA in the brain (Figure 5B). In HCC, HIF proteins play an important role in tumor progression and their expression is a poor prognostic indicator [49], [50]. Therefore to examine the effect of inhibiting SIRT1 on HIF expression and function in HCC, 0.5×106 luciferase-labeled HepG2 cells were injected into the subcapsular space of the left liver lobe in immune deficient Rag2/common gamma- mice. On day 8 after injection, intrahepatic tumors were visible by bioluminescent imaging. Starting on day 9, an i.p. injection of cambinol (100 mg/kg) or vehicle was administered daily, 5 times per week. A preliminary study verified the concentration of cambinol used had no toxic effect to the animals; they displayed no weight loss or increased levels of serum transaminases (ALT & AST) (data not shown). Animals were euthanized on day 30 due to sizeable tumor growth in the vehicle-treated group. Animals treated with cambinol had overall smaller tumors than vehicle treated controls (Figure 5C). Analysis of tumor tissue at time of excision revealed lower mRNA levels of the HIF target gene and pro-angiogenesis factor, VEGF in cambinol treated mice (Figure 5D). Histological examination of the tumors of cambinol treated animals showed less vascular density and intratumoral hemorrhage (Figure 5E). Taken together, these in vivo observations support our in vitro data and further demonstrate that loss of SIRT1 activity impairs HIF-mediated responses to hypoxia.


Inhibition of SIRT1 impairs the accumulation and transcriptional activity of HIF-1α protein under hypoxic conditions.

Laemmle A, Lechleiter A, Roh V, Schwarz C, Portmann S, Furer C, Keogh A, Tschan MP, Candinas D, Vorburger SA, Stroka D - PLoS ONE (2012)

Inhibition of SIRT1 with cambinol impairs hypoxic response in vivo.A. HepG2 cells were treated with 50 and 100 µM sirtinol or cambinol or an equivalent concentration of DMSO (0) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. A representative blot of 2 independently performed experiments is shown. B. Wild type C57BL/6 mice were administered 100 mg/kg cambinol 2 hours prior to exposure to 6% O2 for 6 hours. Total RNA was isolated from liver, kidney and brain tissues. RT-qPCR was performed and ΔCt values of EPO mRNA of hypoxic mice treated with vehicle or cambinol were compared the average delta Ct values of vehicle-treated normoxic mice. The fold change of EPO mRNA was from 221±65 (control) to 88±48 (cambinol), **p = 0.0014 in the kidney and from 120±43 (control) to 21±10.6 (cambinol), *p = 0.01 in the liver and from 9.6±2.7 (control) to 8.3±2.3 (cambinol), p = 0.5036 in the brain. Columns, mean values from 4 independent experiments ±SD. C. Relative luciferase units (RLU) were measured as an indicator of tumor size in mice harboring intrahepatic luciferase-labeled HepG2 tumors. Mice treated with 100 mg/kg cambinol had an overall smaller tumor volume compared to vehicle treated controls. Dots are representative of individual animals, bars are the mean ±SD. D. Total RNA was isolated from HepG2 tumors. ΔCt values of VEGF mRNA in vehicle treated mice were compared to mice treated with cambinol by RT-qPCR. Dots are representative of individual animals and bars are the mean ±SD. E. Haematoxylin and eosin stain of excised tumors from mice treated with vehicle or cambinol.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316573&req=5

pone-0033433-g005: Inhibition of SIRT1 with cambinol impairs hypoxic response in vivo.A. HepG2 cells were treated with 50 and 100 µM sirtinol or cambinol or an equivalent concentration of DMSO (0) for 16 hours and then exposed to 21% O2 (N) or 1% O2 (H) for 4 hours. A representative blot of 2 independently performed experiments is shown. B. Wild type C57BL/6 mice were administered 100 mg/kg cambinol 2 hours prior to exposure to 6% O2 for 6 hours. Total RNA was isolated from liver, kidney and brain tissues. RT-qPCR was performed and ΔCt values of EPO mRNA of hypoxic mice treated with vehicle or cambinol were compared the average delta Ct values of vehicle-treated normoxic mice. The fold change of EPO mRNA was from 221±65 (control) to 88±48 (cambinol), **p = 0.0014 in the kidney and from 120±43 (control) to 21±10.6 (cambinol), *p = 0.01 in the liver and from 9.6±2.7 (control) to 8.3±2.3 (cambinol), p = 0.5036 in the brain. Columns, mean values from 4 independent experiments ±SD. C. Relative luciferase units (RLU) were measured as an indicator of tumor size in mice harboring intrahepatic luciferase-labeled HepG2 tumors. Mice treated with 100 mg/kg cambinol had an overall smaller tumor volume compared to vehicle treated controls. Dots are representative of individual animals, bars are the mean ±SD. D. Total RNA was isolated from HepG2 tumors. ΔCt values of VEGF mRNA in vehicle treated mice were compared to mice treated with cambinol by RT-qPCR. Dots are representative of individual animals and bars are the mean ±SD. E. Haematoxylin and eosin stain of excised tumors from mice treated with vehicle or cambinol.
Mentions: We next tested if pharmacologically targeting SIRT1 can also influence HIF activity in vivo. Cambinol, a cell permeable β-naphthol compound inhibits the NAD+-dependent deacetylase activity of SIRT1 and SIRT2 (IC50 = 56 µM and 59 µM, respectively) and exhibits no inhibition against class I or II histone deacetylase activity [47]. Unlike sirtinol, cambinol can be used in vivo and was shown to effectively inhibit xenograft BCL6-expressing Burkitt lymphoma growth in mice [47]. In a first step, the suppressive effect of cambinol was tested and compared to sirtinol in HepG2 cells in vitro (Figure 5A). HepG2 cells are tumorigenic in immune deficient mice, therefore provide the opportunity to test the effect of SIRT1 inhibition in an HCC xenograft model. Inhibition of SIRT activity with cambinol led to a dose-dependent repression of HIF-1α protein accumulation in HepG2 cells in vitro. Cells treated with sirtinol were used for comparison (Figure 5A). We previously reported that in mice exposed to 6% oxygen HIF-1α protein accumulates in various tissues and activates HIF target genes [48]. Therefore, we tested if inhibition of SIRT1 represses a HIF-driven response in vivo. Mice were pre-treated with cambinol for 2 hours and then exposed to 6% oxygen for 6 hours. Analysis of mouse tissues showed that there was a significant decrease of EPO mRNA in the kidney and the liver in mice pre-treated with cambinol, whereas, pre-treatment with cambinol did not reduce EPO mRNA in the brain (Figure 5B). In HCC, HIF proteins play an important role in tumor progression and their expression is a poor prognostic indicator [49], [50]. Therefore to examine the effect of inhibiting SIRT1 on HIF expression and function in HCC, 0.5×106 luciferase-labeled HepG2 cells were injected into the subcapsular space of the left liver lobe in immune deficient Rag2/common gamma- mice. On day 8 after injection, intrahepatic tumors were visible by bioluminescent imaging. Starting on day 9, an i.p. injection of cambinol (100 mg/kg) or vehicle was administered daily, 5 times per week. A preliminary study verified the concentration of cambinol used had no toxic effect to the animals; they displayed no weight loss or increased levels of serum transaminases (ALT & AST) (data not shown). Animals were euthanized on day 30 due to sizeable tumor growth in the vehicle-treated group. Animals treated with cambinol had overall smaller tumors than vehicle treated controls (Figure 5C). Analysis of tumor tissue at time of excision revealed lower mRNA levels of the HIF target gene and pro-angiogenesis factor, VEGF in cambinol treated mice (Figure 5D). Histological examination of the tumors of cambinol treated animals showed less vascular density and intratumoral hemorrhage (Figure 5E). Taken together, these in vivo observations support our in vitro data and further demonstrate that loss of SIRT1 activity impairs HIF-mediated responses to hypoxia.

Bottom Line: Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2.Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes.In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α.

View Article: PubMed Central - PubMed

Affiliation: Clinic of Visceral Surgery and Medicine, Visceral and Transplantation Surgery, University Hospital Bern and University of Bern, Bern, Switzerland.

ABSTRACT
Sirtuins and hypoxia-inducible transcription factors (HIF) have well-established roles in regulating cellular responses to metabolic and oxidative stress. Recent reports have linked these two protein families by demonstrating that sirtuins can regulate the activity of HIF-1 and HIF-2. Here we investigated the role of SIRT1, a NAD+-dependent deacetylase, in the regulation of HIF-1 activity in hypoxic conditions. Our results show that in hepatocellular carcinoma (HCC) cell lines, hypoxia did not alter SIRT1 mRNA or protein expression, whereas it predictably led to the accumulation of HIF-1α and the up-regulation of its target genes. In hypoxic models in vitro and in in vivo models of systemic hypoxia and xenograft tumor growth, knockdown of SIRT1 protein with shRNA or inhibition of its activity with small molecule inhibitors impaired the accumulation of HIF-1α protein and the transcriptional increase of its target genes. In addition, endogenous SIRT1 and HIF-1α proteins co-immunoprecipitated and loss of SIRT1 activity led to a hyperacetylation of HIF-1α. Taken together, our data suggest that HIF-1α and SIRT1 proteins interact in HCC cells and that HIF-1α is a target of SIRT1 deacetylase activity. Moreover, SIRT1 is necessary for HIF-1α protein accumulation and activation of HIF-1 target genes under hypoxic conditions.

Show MeSH
Related in: MedlinePlus