Inhibition of SIRT1 Impairs the Accumulation and Transcriptional Activity of HIF-1α Protein under Hypoxic Conditions

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

[Surgical proctology - what is common is common]

Proktologische Erkrankungen sind ausgesprochen häufig und werden von den Patienten oft verdrängt oder aus Scham verschwiegen und von den Chirurgen leider auch meist vernachlässigt. Dies mag damit zusammenhängen, dass die Genese dieser Krankheitsbilder völlig unterschiedlich sein kann und von neurologischen Störungen über embryonale Fehlentwicklungen bis zu traumatisch bedingten Defekten reichen kann. Wohl aus diesem Grund wurde in der Vergangenheit die Proktologie von unterschiedlichen Disziplinen mit uneinheitlichen Therapieansätzen und nicht standardisierten Methoden angegangen. Die Interdisziplinarität ist in diesem Bereich der Medizin aber von äusserster Wichtigkeit. Denn nur durch ein gutes Zusammenspiel der verschiedenen Disziplinen in der Diagnostik und Therapie kann das bestmögliche Ergebnis für unsere Patienten erzielt werden. Das höchste Gut für das Leben der Betroffenen und einer der wichtigsten Faktoren in der Therapie ist der Erhalt der Kontinenz. Im Folgenden wollen wir hier auf die häufigsten Krankheitsbilder und deren chirurgische Therapieansätze eingehen.Proctological diseases are extremely common and are often suppressed or concealed by the patients out of shame and unfortunately mostly neglected by the surgeons. This may have to do with the fact that the genesis of these clinical pictures can be completely different and can range from neurological disorders to embryonic maldevelopment to traumatically caused defects. Probably for this reason, in the past proctology was approached by different disciplines with inconsistent therapeutic approaches and non-standardized methods. However, interdisciplinarity is of utmost importance in this field of medicine. Only through a good interaction of the different disciplines in diagnostics and therapy can the best possible outcome be achieved for our patients. The highest good for the life of those affected and one of the most important factors in therapy is the preservation of continence

How do we apply video-assisted retroperitoneal necrosectomy with minimal access?

The conservative treatment of acute necrotizing pancreatitis has greatly improved due to broad antibiotic treatment and improved organ support in intensive care units. Nevertheless, infected necrosis or persistent multi-organ dysfunction are predictors of poor outcome. In these patients, there is still a need to perform necrosectomy. Open surgery results in extensive operative trauma and is associated with high morbidity and mortality. Therefore, several minimally invasive techniques have been developed recently. Retroperitoneal necrosectomy has been shown to be safe and to reduce morbidity and mortality compared to the open procedure

Antitumor effect of SIRT1 inhibition in human HCC tumor models in vitro and in vivo

Sirtuins (SIRT1-7) are a highly conserved family of NAD(+)-dependent enzymes that control the activity of histone and nonhistone regulatory proteins. SIRT1 is purposed to promote longevity and to suppress the initiation of some cancers. Nevertheless, SIRT1 is reported to function as a tumor suppressor as well as an oncogenic protein. Our data show that compared with normal liver or surrounding tumor tissue, SIRT1 is strongly overexpressed in human hepatocellular carcinoma (HCC). In addition, human HCC cell lines (Hep3B, HepG2, HuH7, HLE, HLF, HepKK1, skHep1) were screened for the expression of the sirtuin family members and only SIRT1 was consistently overexpressed compared with normal hepatocytes. To determine its effect on HCC growth, SIRT1 activity was inhibited either with lentiviruses expressing short hairpin RNAs or with the small molecule inhibitor, cambinol. Knockdown or inhibition of SIRT1 activity had a cytostatic effect, characterized by an altered morphology, impaired proliferation, an increased expression of differentiation markers, and cellular senescence. In an orthotopic xenograft model, knockdown of SIRT1 resulted in 50% fewer animals developing tumors and cambinol treatment resulted in an overall lower tumor burden. Taken together, our data show that inhibition of SIRT1 in HCC cells impairs their proliferation in vitro and tumor formation in vivo. These data suggest that SIRT1 expression positively influences the growth of HCC and support further studies aimed to block its activity alone or in combination as a novel treatment strategy

SIRT1 inhibition represses HIF-1 transcriptional activity and HIF-1α protein.

<p><b>A.</b> Hep3B cells were co-transfected with luciferase reporter carrying multiple HREs and a renilla luciferase control plasmid. Twenty-four hours after the transfection, cells were treated with 25, 50 and 100 µM sirtinol and exposed to hypoxia for 24 hours. Dual luciferase activities were measured and firefly values were normalized by renilla values. Luciferase activity was reduced from 35-fold of DMSO-treated controls to 32-fold with 25 µM sirtinol, p = 0.2611; to 23-fold with 50 µM, *p = 0.0225 and to 10.5-fold with 100 µM ***p = 0.0009. <i>Columns</i>, mean of triplicates from one representative experiment (n = 3, independent experiments) <i>Bars</i>, ±SD. <b>B.</b> Hep3B cells were either treated with 100 µM sirtinol or an equivalent concentration of DMSO for 4 hours and then exposed to 1% O₂ for 16 hours. Fold increase was calculated from ΔCt values of hypoxic cells to average ΔCt values of cells cultured at 21% O₂ in DMSO. BNIP3 mRNA was reduced from 14±1.5 to 3.4±1-fold (***p = 0.0005), CA9 mRNA from 21±3.5 to 3.8±0.8-fold (**p = 0.0011) and EPO from 100±11.5 to 6.1±1.6-fold (**p = 0.0012) <i>Columns</i>, mean of 3 independent experiments; <i>bars</i>, ±SD. <b>C.</b> Hep3B cells were infected with lentiviral vectors containing shRNA sequences that target SIRT1 (shSIRT1_1958) or with a scrambled control (SHC002). Five days after transduction cells were exposed to hypoxia for 12 hours. The relative fold increase of mRNA in hypoxic cells was calculated compared to normoxic controls. CA9 mRNA reduced from 27- to 5.5-fold (**p = 0.008) and EPO from 120- to 18-fold (**p = 0.004). <i>Columns</i>, mean of 3 independent experiments; <i>bars</i>, ±SD. <b>D.</b> Hep3B cells were treated with 0, 25, 50 and 100 µM sirtinol or an equivalent concentration of DMSO (D) for 16 hours and then exposed to 21% O₂ (N) or 1% O₂ (H) for 4 hours. Whole cell lysates were analyzed by Western blot using antibodies against HIF-1α. α-tubulin was used as a loading control. A representative blot of 6 independently performed experiments is shown. <b>E.</b> Hep3B cells were infected with lentiviral vectors containing shRNAs targeting SIRT1 (shSIRT1_1958 and shSIRT1_3206) or with a scrambled negative control (SHC002). Five days after transduction, cells were exposed to hypoxia for 4 hours and SIRT1 and HIF-1α were analyzed by Western blot. Representative blot of 3 independently performed experiments. <b>F–G.</b> Hep3B cells were treated with 100 µM sirtinol for 16, 2 and 0 hours before and 2 hours after the exposure to hypoxia for a total of 4 hours. HIF-1α expression was analyzed by Western blot and by RT-qPCR. <b>H.</b> RCC4 VHL+/+ and RCC4 VHL−/− cells were pretreated with 100 µM sirtinol or DMSO for 2 or 16 hours, followed by 4 hours exposure to 21% O₂ (N) or 1% O₂ (H). Whole cell lysates were analyzed for HIF-1α by Western blot.</p

Inhibition of SIRT1 with cambinol impairs hypoxic response <i>in vivo</i>.

<p><b>A.</b> 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>B.</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. <i>Columns</i>, mean values from 4 independent experiments ±SD. <b>C.</b> 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. <b>D.</b> 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. <b>E.</b> Haematoxylin and eosin stain of excised tumors from mice treated with vehicle or cambinol.</p

SIRT1 overexpression stabilizes HIF-1α protein.

<p>Hep3B cells were infected with lentiviruses expressing either SIRT1wt or mutated SIRT1 H363Y. Three days after transduction clones were selected and expanded. Hep3B cells with SIRT1wt clone 5 and clone 13 or SIRT1 H363Y lines were incubated with 0, 20 and 50 µM DMOG for 2 hours. Whole cell lysates were analyzed for SIRT1 and HIF-1α by Western blot. A representative blot of 3 independently performed experiments is shown.</p

SIRT1 and HIF-1α co-immunoprecipitate.

<p><b>A.</b> Hep3B cells were treated with 125 µM DMOG for 5 hours. Cytoplasmic and nuclear protein fractions were prepared. Twenty-five µg nuclear proteins and 50 µg cytoplasmic proteins were analyzed for SIRT1 and HIF-1α by Western blot. Sp1 was used as a nuclear fraction marker and α-tubulin for the cytoplasmic fraction. A representative blot of 3 independently performed experiments is shown. <b>B.</b> Cytoplasmic proteins (1250 µg) prepared in (A) were immunoprecipitated with a rabbit polyclonal SIRT1 antibody, chicken polyclonal HIF-1α antibody or a rabbit IgG1 control antibody. Immunoprecipitated proteins were subjected to Western blot analysis. A representative blot of 3 independently performed experiments is shown. <b>C.</b> SIRT1 inhibition leads to increased acetylation of HIF-1α. Hep3B cells were infected with lentiviral vectors containing shRNA against SIRT1 (shSIRT1_1958 or shSIRT1_3206) or scrambled shRNA (SHC002) as a negative control. Seven days post-infection, cells were incubated under hypoxia for 5 hours in the presence of 10 µM MG132. Whole cell lysates (2 mg) were immunoprecipitated with a mouse monoclonal HIF-1α antibody (2 µg) and precipitates were analyzed by Western blotting with a rabbit polyclonal anti-acetyl-lysine and a polyclonal chicken anti-HIF-1α antibody. Whole cell lysates (WCL: 100 µg) were used as input controls and were analyzed for SIRT1 and α-tubulin. A representative blot of 2 independently performed experiments is shown.</p