Effects of S100A7 overexpression on mitochondrial dynamics and autophagy activation.

<p>S100A7-HaCaT cells were treated for 24 h with LPS at 100 and 1000 ng/ml. (<b>A</b>, <b>B</b>) The mitochondrial dynamics-related proteins Mfn1, Mfn2 and DRP1 and (<b>C</b>, <b>D</b>) the autophagy-related proteins Beclin1 and LC3B were detected by Western blot (<b>A</b>, <b>C</b> western blot images; <b>B</b>, <b>D</b> statistical results). Values are means ± SEM. Statistical significance is indicated (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ^#<i>P</i><0.05, ∧∧<i>P</i><0.01, ∧∧∧<i>P</i><0.001). veh, vehicle; veh-100 or veh-1000, vehicle with 100 or 1000 ng/ml LPS treatment; pso, S100A7; pso-100 or pso-1000, S100A7-EGFP HaCaT cells with 100 or 1000 ng/ml LPS treatment.</p

Characterization of S100A7 overexpression in HaCaT cells.

<p>(<b>A</b>) S100A7 mRNA levels in HaCaT cells overexpressing S100A7. S100A7 mRNA is normalized to β-actin. The data represent the mean ± SEM of three independent experiments. Statistical significance (**<i>P</i><0.01, ^##<i>P</i><0.01). (<b>B</b>) Immunofluorescence analysis of S100A7 overexpression. Cells were stained for EGFP (green) and anti-S100A7 (red); the nuclei were stained with DAPI (blue). Veh, vehicle; Veh-EGFP, vehicle-EGFP; pso-EGFP, S100A7-EGFP.</p

Effects of S100A7 overexpression on the LPS-induced mRNA and protein expression of IL-6 and IL-8.

<p>(<b>A</b>) IL-6 and IL-8 mRNA levels and (<b>B</b>) protein levels were assayed after stable HaCaT cells (S100A7-EGFP or vehicle-EGFP) were exposed to LPS (100 or 1000 ng/ml, 24 h). The data represent the means ± SEM of three independent experiments. Statistical significance is indicated (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). veh, vehicle; veh-100 or veh-1000, vehicle with 100 or 1000 ng/ml LPS treatment; pso, S100A7; pso-100 or pso-1000, S100A7-EGFP HaCaT cells with 100 or 1000 ng/ml LPS treatment.</p

Effects of S100A7 overexpression on the expression of mitochondrial biogenesis and function.

<p>S100A7-EGFP and vehicle-EGFP HaCaT cells were exposed to lipopolysaccharide (LPS) (100 or 1000 ng/ml, 24 h). (<b>A</b>, <b>B</b>, <b>C</b>) The mRNA levels of PPAR-coactivator-1alpha (PGC-1α), nuclear respiratory factor-1 (NRF1) and transcription factor A (Tfam) were analyzed by quantitative RT-PCR. (<b>D</b>, <b>E</b>, <b>F</b>, <b>G</b>) Relative mitochondrial DNA contents (mtDNA), mitochondrial membrane potential (MMP), intracellular intracellular adenosine 5′-triphosphate (ATP) level and reactive oxygen species (ROS) generation were measured respectively. Values are means ± SEM; *Statistical significance (*<i>P</i><0.05, **<i>P</i><0.01). veh, vehicle; veh-100 or veh-1000, vehicle with 100 or 1000 ng/ml LPS treatment; pso, S100A7; pso-100 or pso-1000, S100A7-EGFP HaCaT cells with 100 or 1000 ng/ml LPS treatment.</p

Effects of S100A7 knockdown on MMP and mitochondrial homeostasis.

<p>(<b>A</b>) Expression of S100A7 mRNA after siRNA transfection. HaCaT cells were transfected with S100A7 siRNA using lipofectamin 2000 for 48 h. Total RNA was extracted and subjected to quantitative RT-PCR. The amount of S100A7 mRNA relative to β-actin is shown. (<b>B</b>) S100A7-siRNA HaCaT cells were treated for 24 h with LPS at 100 and 1000 ng/ml. The MMPs were assayed. (<b>C</b>, <b>D</b>) The mitochondrial dynamics-related proteins DRP1 and Mfn2 and the autophagy-related protein LC3B were detected by Western blot (<b>C</b>, Western blot images; <b>D</b>, statistical results). Values are means ± SEM. Statistical significance is indicated (^#<i>P</i><0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001). cont, control; veh-si, vehicle-siRNA; veh-si-100 or veh-si-1000, vehicle-siRNA cells with 100 or 1000 ng/ml LPS treatment; pso-si, S100A7-siRNA; pso-si-100 or pso-si-1000, S100A7-siRNA HaCaT cells with 100 or 1000 ng/ml LPS treatment.</p

SIRT3 Enhances Glycolysis and Proliferation in SIRT3-Expressing Gastric Cancer Cells

<div><p>SIRT3 is a key NAD⁺-dependent protein deacetylase in the mitochondria of mammalian cells, functioning to prevent cell aging and transformation via regulation of mitochondrial metabolic homeostasis. However, SIRT3 is also found to express in some human tumors; its role in these SIRT3-expressing tumor cells needs to be elucidated. This study demonstrated that the expression of SIRT3 was elevated in a group of gastric cancer cells compared to normal gastric epithelial cells. Although SIRT3 expression levels were increased in the gastric tumor tissues compared to the adjacent non-tumor tissues, SIRT3 positive cancer cells were more frequently detected in the intestinal type gastric cancers than the diffuse type gastric cancers, indicating that SIRT3 is linked with subtypes of gastric cancer. Overexpression of SIRT3 promoted cell proliferation and enhanced ATP generation, glucose uptake, glycogen formation, MnSOD activity and lactate production, which were inhibited by SIRT3 knockdown, indicating that SIRT3 plays a role in reprogramming the bioenergetics in gastric tumor cells. Further analysis revealed that SIRT3 interacted with and deacetylated the lactate dehydrogenase A (LDHA), a key protein in regulating anaerobic glycolysis, enhancing LDHA activity. In consistence, a cluster of glycolysis-associated genes was upregulated in the SIRT3-overexpressing gastric tumor cells. Thus, in addition to the well-documented SIRT3-mediated mitochondrial homeostasis in normal cells, SIRT3 may enhance glycolysis and cell proliferation in SIRT3-expressing cancer cells.</p></div

SIRT3 promotes aggressive characteristics of gastric cancer cells.

<p>A, cell growth of AGS and SGC-7901 cells with SIRT3 overexpression or knockdown was calculated on days 2, 4, 6 and 8 after cell plating. SIRT3 expression in stable transfectants was confirmed by western blot. B, clonogenicity of AGS and SGC-7901 cells with SIRT3 overexpression or knockdown was measured and presented as the fraction of control transfectants (NC or Scr). C, Overexpression of SIRT3 promoted tumor burden in vivo. SGC-7901 cells with SIRT3 overexpression (left panel) or knockdown (right panel) were subcutaneously injected into right flank of the nude mice with the relative control cells (NC or Scr) into the left flank. Xenograft tumors were excised and weighed at the 28^th day after cell inoculation. Images of right panel showed xenograft tumors in vivo at the end of the experiment. Images of up right corner showed the dissected tumors from each group. The ranges and means of tumor weights of each group were presented in right panel as mean ± S.E. (n = 5; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01). SIRT3, SIRT3 overexpression; shSIRT3, SIRT3 knockdown; NC (negative control; empty pcDNA3.1) and Scr (scramble shRNA; pGPH1/GFP/Neo-shRNA) serve as controls for pcDNA3.1-SIRT3 and pGPH1/GFP/Neo-shSIRT3 respectively.</p

SIRT3 interacts with LDHA and regulates LDHA activity through deacetylation.

<p>A, LDHA activity was measured in AGS and SGC-7901 cells with SIRT3 overexpression or knockdown and represented as relative activity normalized by control (NC or Scr). LDHA expression in SIRT3 overexpression and knockdown AGS cells was analyzed by immunoblotting. B, co-localization of SIRT3 and LDHA was detected by immunostaining in AGS cells with anti-SIRT3 (green) and anti-LDHA (red) antibodies. Nuclei were conterstained with DAPI (blue). Scale bar, 5 μm. C, LDHA was immunoprecipitated (IP) followed by western blot (WB) of LDHA or SIRT3, or reverse, in AGS gastric cancer cells. IP with non-immune goat IgG serves as a negative control, and immunoblotting of total cell lysates (input) serves as the IP equal loading control. D, SIRT3-enhanced deacetylation of LDHA in AGS cells was detected by IP with anti-LDHA followed by western blot with anti-LDHA and anti-acetyl-lysine antibodies. Western blot of total cell lysates with SIRT3 antibody to show the expression of transfected protein. E, LDHA enzymatic activity was measured using commercial bovine heart L-Lactic dehydrogenase and recombinant human SIRT3 enzyme with/without SIRT3 inhibitor nicotinamide. F, LDHA enzymatic activity was measured using commercial recombinant human SIRT3 enzyme and immunoprecipitated LDHA from AGS cells transfected with K5Q/R or K318Q/R mutant LDHA with/without SIRT3 inhibitor nicotinamide and presented as relative enzyme activity normalized by wild type LDHA without SIRT3 inhibitor. In A, E and F, data are presented as mean ± S.E. (n = 5; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01).</p

Overexpression of SIRT3 enhances glycolysis in gastric cancer cells.

<p>Glucose uptake, lactate production and glycogen formation were measured in AGS (A, C, E) and SGC-7901 (B, D, F) cells with SIRT3 overexpression or knockdown. All data are presented as mean ± S.E. (n = 3; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01).</p

Genes potentially regulated by SIRT3 in gastric cancer cells.

<p>Relative mRNA levels of Glut1, HK2, MCT1 and MCT4 were measured by qRT-PCR in AGS (A) and SGC-7901 (B) cells with SIRT3 overexpression or knockdown. Data are presented as mean ± S.E. (n = 3; *, <i>p</i> < 0.05; **, <i>p</i> < 0.01). (C) Schematic presentation of the potential mechanism by which SIRT3 triggers the cell growth. SIRT3 deacetylates and activates LDHA causing enhanced LDHA activity and cellular bioenergetics, which together with SIRT3-mediated MnSOD activation enhances cell proliferation.</p