A H₂S-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 (H₂S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H₂S-Nampt energetic circuit, in damage recovered cancer cells, H₂S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H₂S 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 H₂S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H₂S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H₂S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H₂S-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 H₂S-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

Changes in glycolysis and bioenergetics in DR cells.

<p>(A) ECAR in Pc HepG2 cells treated with or without 800 µM of H₂O₂ and DR^{H2O2 W2} HepG2 cells. (B) ATP levels in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.005 in ANOVA statistical analysis. (C) NAD⁺ levels in Pc HepG2 (with or without H₂O₂), DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. NAD⁺ was normalized to the level of total protein. Significance between Pc and there DR cells was <i>p</i><0.005 in ANOVA statistical analysis. (D) NAD⁺ levels in Pc and DR^{H W1} HepG2 cells. (E) Western blot analysis of intracellular Nampt and CBS in Pc and DR^H2O2 MDA-MB-231 cells. (F) Nampt levels assessed by ELISA in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.05 in ANOVA statistical analysis. (G) Correlation between Nampt expression and production of H₂S and level of ATP in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance of H₂S and Nampt was <i>p</i><0.05, and significance of ATP and Nampt was <i>p</i><0.05 in ANOVA statistical analysis. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S levels and bioenergetic changes in DR cells isolated from tumors.

<p>(A) H₂S levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (B) Expression of CBS and CTH in T^V and T^DR cells. (C) NAD⁺ levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (D) Nampt levels in T^V and T^DR cells isolated from HepG2 tumors grown <i>in vivo</i>. (E) ECAR in T^V and T^DR 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^V) isolated from HepG2 tumors grown <i>in vivo</i> expressed as the percent of the ATP level in Pc. (G) Proliferation of T^V and T^DR 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 2×10⁴ and the total number of cells was assessed after 24 and 48 hr of culture. (H) A scheme for H₂S-Nampt dependent bioenergetic circuit. Cell damage leads to increased H₂S 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 H₂S and proliferation rate and exhibit tolerance to damage.

<p>(A) A scheme for isolation of Damage-Recovered (DR) cells. (B) Bax expression in H₂O₂ treated Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (C) Amount of H₂S released by DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. Significance between Pc and three DR cells was <i>p</i><0.0005 in ANOVA statistical analysis. (D) H₂S staining of Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells with 5 µM H₂S 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^{H2O2 W1} and DR^{H2O2 W3} HepG2 cells. (F) Western blot analysis of CBS and CTH in Pc, DR^{H2O2 W1}, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells. (G) Western blot analysis of CBS in Pc and DR^{H2O2 W1}, DR^{H W1} and DR^{G W1} HepG2 cells. (H) Proliferation of HepG2 recovered from H₂O₂, DR^{H2O2 W1}, DR^{H2O2 W2} cells as a percentage of that in Pc cells. (I) Viability of Pc, DR^{H2O2 W1} and DR^{H2O2 W2} HepG2 cells with and without treatment with bleomycin. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

Endogenous hydrogen sulfide increases in response to acute damage in cancer cells.

<p>(A) Amount of H₂S released by 293 cells, fibroblasts (Fibro.), HepG2, MDA-MB-231 and MDA-MB-435S cells. (B) The levels of H₂S in HepG2, MDA-MB-231 and MDA-MB-435S Pc cells subjected to hypoxia (0.5% O₂, 18 hr), glucose deprivation (glucose free medium, 18 hr) or treatment with bleomycin (35 nM, 18 hr), H₂O₂ (800 µM, 3 hr). Data are expressed as percent of H₂S 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 H₂O₂ for 3 hr. (D) The level of CBS and γH2AX with or without treatment with H₂O₂ (800 µM, 3 hr) in MDA-MB-435S cells. Protein density was normalized using β-Actin. (E) Amount of H₂S and cell viability after treatment with a range of concentration of H₂O₂. *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S-Nampt pathway regulates bioenergetics.

<p>(A) Amount of H₂S released from DR^{H W1} HepG2 cells treated with CTH inhibitor, PAG (100 µM, 18 hr), and Nampt inhibitor, FK866 (200 nM, 24 hr). (B) Western blot analysis of CBS in DR cells in the absence and presence of FK866 (200 nM, 24 hr). (C) Western blot analysis of CTH in DR cells in the absence and presence of FK866 (200 nM, 24 hr). (D) ATP levels in DR^{H2O2 W3} HepG2 cells in the absence (−) and presence (+) of PAG (100 µM, 18 hr). (E) ATP levels in Pc, DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells in the absence and presence of FK866 (200 nM, 24 hr). (F) Western blot analysis of Nampt in DR^{H2O2 W2} and DR^{H2O2 W3} HepG2 cells treated with CBS inhibitor, CHH (500 µM, 18 hr) or CTH inhibitor, PAG (100 µM, 18 hr). *; <i>p</i><0.05,**; <i>p</i><0.005, ***; <i>p</i><0.0005.</p

H₂S increases ECAR, ATP, NAD⁺ 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^{H W1} HepG2 cells. (B) Comparison of ATP levels in Pc HepG2 cells treated for 48 hr with 0, 10 and 100 µM of NaHS, DR^{H2O2 W1} 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⁺ 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 H₂O₂ (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