12 research outputs found
NaHS-treatment increases expression of <i>NAMPT</i> and <i>SIRT1</i>.
<p>(<b>A</b> and <b>B</b>) The expression of <i>NAMPT</i> and <i>SIRT1</i> in young (PD: 5.9) and senescent (PD: 18.8) was assessed by real-time PCR and normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) Immunoblotting of Nampt and Sirt1 in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. β-Actin was used as a loading control. (<b>D</b> and <b>E</b>) Young (PD: 5.9) aHDF cells were treated without and with NaHS for 3 days, and RNA samples were then subjected to real-time PCR for assessment of <i>NAMPT</i> and <i>SIRT1</i>. The expression levels of <i>NAMPT</i> and <i>SIRT1</i> were normalized to the levels of expression of <i>β-ACTIN</i>. (<b>F</b>) Immunoblotting of Nampt and Sirt1 in NaHS-treated young (PD: 5.9) aHDF cells. β-Actin was used as a loading control. (<b>G</b>) NAD/NADH ratio in young (PD: 5.9) aHDF cells treated without and with NaHS for 7 days. Data were normalized to the total amount of protein.</p
Exogenous H<sub>2</sub>S increases PD and suppresses SA-β-Gal expression.
<p>(<b>A</b>) PD of cells treated without or with 1 μM NaHS. The population doubling of the first confluent cultures was designated as 0. (<b>B</b>) Representative images of SA–β-Gal staining in cells shown in Fig 3A. Mean values ± error bars of number of SA-β-Gal positive cells are shown on the right-upper corner of each image. *; p<0.05, ***; p<0.0005, n.s.; not significant. Scale bars, 100 μm.</p
Production of H<sub>2</sub>S is downregulated in replicatively senescent cells.
<p>(<b>A</b>) Representative images of SA-β-Gal staining in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Scale bars, 100 μm. (<b>B</b>) Real-time PCR analysis of expression of <i>hTERT</i> in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>hTERT</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>C</b>) NAD/NADH ratio in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. Real-time PCR analysis of expression of <i>CBS</i> (<b>D</b>), <i>MST</i> (<b>E</b>), and <i>CSE</i> (<b>F</b>) in young (PD: 5.9) and senescent (PD: 18.8) aHDF cells. The expression of <i>CBS</i>, <i>MST</i>, and <i>CSE</i> was normalized to the expression level of <i>β-ACTIN</i>. (<b>G</b>) 1 x 10<sup>6</sup> cells of young (PD: 5.9) and senescent (PD: 18.8) aHDF cells were incubated in PBS at 37°C for 1 hour and then H<sub>2</sub>S was measured in culture supernatants. Mean values are shown along with error bars. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p
H<sub>2</sub>S induces <i>hTERT</i> expression in a NAMPT/SIRT1-dependent manner.
<p>(<b>A</b> and <b>B</b>) Downregulation of <i>SIRT1</i> suppresses the expression of <i>hTERT</i>. Young (PD: 5.9) aHDF cells (3 x 10<sup>5</sup> cells) were transfected with <i>SIRT1</i> siRNA for 2 days, and these were treated without or with NaHS for 3 days. Total RNAs from these cells were subjected to real-time PCR analysis for <i>SIRT1</i> (<b>A</b>) and <i>hTERT</i> (<b>B</b>). Data were normalized to the level of expression of <i>β-ACTIN</i>. The expression level of <i>SIRT1</i> and <i>hTERT</i> in cells treated with Scrambled siRNA without NaHS treatment was regarded as 1.0. (<b>C</b>) Downregulation of <i>NAMPT</i> suppresses the activity of SIRT1. Young (PD: 5.9) aHDF cells (3 x 10<sup>5</sup> cells) were transfected with <i>NAMPT</i> siRNA for 2 days, and then the cells were treated without or with 1 μM NaHS for 3 days. Nuclear proteins were extracted and used for measurement of SIRT1 activity. Mean values ± error bars were normalized to the amount of total cell protein. (<b>D</b>) Mode of action of H<sub>2</sub>S in opposing senescence. *; <i>p</i><0.05, **; <i>p</i><0.005, ***; <i>p</i><0.0005, n.s.; not significant.</p
Exogenous H<sub>2</sub>S increases the expression of <i>hTERT</i> as well as the activity of telomerase.
<p>(<b>A</b>) Real-time PCR analysis of the expression of <i>hTERT</i> in young (PD: 5.9) aHDF cells, treated with NaHS for 3 days. The expression of <i>hTERT</i> was normalized to the level of expression of <i>β-ACTIN</i>. Expression of untreated control was regarded as 1.0. (<b>B</b>) Immunoblotting of hTERT in aHDF cells without or with 1 μM NaHS for 7 days. 100 μg of the indicated nuclear extracts were subjected for immunoblotting. β-Actin was used as a loading control. (<b>C</b>) Telomerase activity in young (PD: 3.2) aHDF cells without or with treated with 1 μM NaHS for 7 days. Positive control was MDA-MB-231 cell lysate, and negative control was buffer alone. Bottom panel shows quantified means ± error bars from three independent assays. Relative activity of telomerase was calculated by dividing the density of all ladders to the density of the bands in internal control, indicated as internal control (I.C.).</p
Endogenous hydrogen sulfide increases in response to acute damage in cancer cells.
<p>(A) Amount of H<sub>2</sub>S released by 293 cells, fibroblasts (Fibro.), HepG2, MDA-MB-231 and MDA-MB-435S cells. (B) The levels of H<sub>2</sub>S in HepG2, MDA-MB-231 and MDA-MB-435S Pc cells subjected to hypoxia (0.5% O<sub>2</sub>, 18 hr), glucose deprivation (glucose free medium, 18 hr) or treatment with bleomycin (35 nM, 18 hr), H<sub>2</sub>O<sub>2</sub> (800 µM, 3 hr). Data are expressed as percent of H<sub>2</sub>S released from untreated cells. (C) Intracellular CBS and Bax (left panel) assessed by western blot analysis in MDA-MB-435S Pc cells, and Pc cells treated with 400 or 800 µM of H<sub>2</sub>O<sub>2</sub> for 3 hr. (D) The level of CBS and γH2AX with or without treatment with H<sub>2</sub>O<sub>2</sub> (800 µM, 3 hr) in MDA-MB-435S cells. Protein density was normalized using β-Actin. (E) Amount of H<sub>2</sub>S and cell viability after treatment with a range of concentration of H<sub>2</sub>O<sub>2</sub>. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p
A H<sub>2</sub>S-Nampt Dependent Energetic Circuit Is Critical to Survival and Cytoprotection from Damage in Cancer Cells
<div><p>We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H<sub>2</sub>S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H<sub>2</sub>S-Nampt energetic circuit, in damage recovered cancer cells, H<sub>2</sub>S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H<sub>2</sub>S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H<sub>2</sub>S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H<sub>2</sub>S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H<sub>2</sub>S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H<sub>2</sub>S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H<sub>2</sub>S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer.</p></div
H<sub>2</sub>S levels and bioenergetic changes in DR cells isolated from tumors.
<p>(A) H<sub>2</sub>S levels in T<sup>V</sup> and T<sup>DR</sup> cells isolated from HepG2 tumors grown <i>in vivo</i>. (B) Expression of CBS and CTH in T<sup>V</sup> and T<sup>DR</sup> cells. (C) NAD<sup>+</sup> levels in T<sup>V</sup> and T<sup>DR</sup> cells isolated from HepG2 tumors grown <i>in vivo</i>. (D) Nampt levels in T<sup>V</sup> and T<sup>DR</sup> cells isolated from HepG2 tumors grown <i>in vivo</i>. (E) ECAR in T<sup>V</sup> and T<sup>DR</sup> cells isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the ECAR level in Pc. (F) ATP level in viable tumor cells (T<sup>V</sup>) isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the ATP level in Pc. (G) Proliferation of T<sup>V</sup> and T<sup>DR</sup> cells isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the proliferation of Pc. Cells were seeded at a concentration of 2×10<sup>4</sup> and the total number of cells was assessed after 24 and 48 hr of culture. (H) A scheme for H<sub>2</sub>S-Nampt dependent bioenergetic circuit. Cell damage leads to increased H<sub>2</sub>S and Nampt that coordinately lead to metabolic changes. These cells exhibit exponential growth and tolerance to damage. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p
Damage-Recovered (DR) cells show increase in H<sub>2</sub>S and proliferation rate and exhibit tolerance to damage.
<p>(A) A scheme for isolation of Damage-Recovered (DR) cells. (B) Bax expression in H<sub>2</sub>O<sub>2</sub> treated Pc, DR<sup>H2O2 W1</sup>, DR<sup>H2O2 W2</sup> and DR<sup>H2O2 W3</sup> HepG2 cells. (C) Amount of H<sub>2</sub>S released by DR<sup>H2O2 W1</sup>, DR<sup>H2O2 W2</sup> and DR<sup>H2O2 W3</sup> HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.0005 in ANOVA statistical analysis. (D) H<sub>2</sub>S staining of Pc, DR<sup>H2O2 W1</sup>, DR<sup>H2O2 W2</sup> and DR<sup>H2O2 W3</sup> HepG2 cells with 5 µM H<sub>2</sub>S fluorescent probe, HSN2. Scale bars, 50 µm. (E) PCR analysis of <i>CBS</i>, <i>CTH</i> and <i>MTS</i> genes in Pc, DR<sup>H2O2 W1</sup> and DR<sup>H2O2 W3</sup> HepG2 cells. (F) Western blot analysis of CBS and CTH in Pc, DR<sup>H2O2 W1</sup>, DR<sup>H2O2 W2</sup> and DR<sup>H2O2 W3</sup> HepG2 cells. (G) Western blot analysis of CBS in Pc and DR<sup>H2O2 W1</sup>, DR<sup>H W1</sup> and DR<sup>G W1</sup> HepG2 cells. (H) Proliferation of HepG2 recovered from H<sub>2</sub>O<sub>2</sub>, DR<sup>H2O2 W1</sup>, DR<sup>H2O2 W2</sup> cells as a percentage of that in Pc cells. (I) Viability of Pc, DR<sup>H2O2 W1</sup> and DR<sup>H2O2 W2</sup> HepG2 cells with and without treatment with bleomycin. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p
H<sub>2</sub>S increases ECAR, ATP, NAD<sup>+</sup> and Nampt in a dose-dependent manner in cancer cells.
<p>(A) ECAR in Pc HepG2 cells treated for 48 hr with 0, 1, and 100 µM of NaHS and DR<sup>H W1</sup> HepG2 cells. (B) Comparison of ATP levels in Pc HepG2 cells treated for 48 hr with 0, 10 and 100 µM of NaHS, DR<sup>H2O2 W1</sup> HepG2 cells and Pc MDA-MB-231 cells treated for 48 hr with 0, 10 and 100 µM of NaHS. Data are expressed as a percent of level of ATP in untreated cells. (C) Levels of NAD<sup>+</sup> in HepG2 and MDA-MB-231 Pc cells treated with 0, 10, and 100 µM of NaHS for 48 hr. (D) Western blot analysis of intracellular Nampt in MDA-MB-231 Pc cells treated for 48 hr with 0 and 100 µM of NaHS. (E) qPCR analysis of <i>NAMPT</i> expression in Pc HepG2 cells treated for 48 hr with 0 and 100 µM of NaHS. (F) Quantitation of intracellular Nampt by ELISA in Pc HepG2 cells treated for 48 hr with 0, 10 and 100 µM of NaHS. (G) Viability in HepG2 cells pre-treated for 24 hr with 0, 1, and 10 mM of NaHS and then subjected to H<sub>2</sub>O<sub>2</sub> (800 µM) or bleomycin (35 nM, 18 hr). Viability was assessed by Trypan blue exclusion. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p