PCA and PLS-DA for MCF-7, MCF-7/shCK-α, MCF-7/TAM and MCF-7/TAM/shCK-α groups.

<p>(A) PCA for the inspection and overview of the data set. (B) Score plot for the investigation of group differentiation among MCF-7 (MC, n = 4, green), MCF-7/shCK-α (Msh, n = 6, blue), MCF-7/TAM (MT, n = 4, red), and MCF-7/TMA/shCK-α (MTsh, n = 5, yellow) cell groups. (C) VIP values as a measure of the discriminatory potential of the individual metabolites in the group differentiation. (UA: unknown resonance A, lactate, UE: unknown resonance E, glutamine, formate, AXP: AMP/ADP/ATP, UXP: UMP/UDP/UTP, MAU: maleate/AXP/UXP, UC: unknown resonance C, NAG: N-acetyl-aspartate/-amino acid/glutamate, Glx: glutamate/glutamine, LIV: leucine/isoleucine/valine). (D) Loading plot showing the relative contribution of the metabolites to the scores in the score plot. By matching the positions in the score plot and loading plot, the correlations between the individual metabolites and the cell groups were determined.</p

Comparison of the concentrations of the metabolites with VIP>1 among the MCF-7, MCF-7/shCK-α, MCF-7/TAM, and MCF-7/TAM/shCK-α cell groups.

<p>Increased lactate, glycine, phosphocholine, glutamine, and succinate in MCF-7/TAM relative to the other cell groups. Increased myo-inositol in MCF-7/shCK-α relative to the other cells. Non-detectable formate in MCF-7/TAM and MCF-7/TAM/shCK-α. Non-detectable fumarate in 7/shCK-α and MCF-7/TAM and MCF-7/TAM/shCK-α. Decreased AXP in MCF-7/shCK-α cells and MCF-7/TAM/shCK-α relative to MCF-7 and MCF-7/TAM. The spectra were normalized against total intensity and averaged over the samples in MCF-7, MCF-7/shCK-α, MCF-7/TAM and MCF-7/TAM/shCK-α. All values are presented as the mean ± standard error. *<i>p</i><0.05, ** 0.001<i><0.05, *** <i>p</i><0.001.</i></p

Assignment of metabolites observed by ¹H-NMR in cancer cell lysates.

<p>Assignment of metabolites observed by ¹H-NMR in cancer cell lysates.</p

Reduction of myc-Nkx2.5 protein in cells treated with <i>O</i>-GlcNAcase inhibotirs.

<p>(A) Immunoblotting of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transfected HEK293 cells in the presence or absence of 3 mM streptozotocine (STZ). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in STZ-treated and untreated cells. <i>O</i>-GlcNAcylation levels were compared in untreated cells <i>vs</i> STZ-treated cells and untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> STZ-treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05. (C) Immunoblotting analysis of myc-Nkx2.5 and <i>O</i>-GlycNAcylated proteins in myc-Nkx2.5-transduced HEK293 cells in the presence or absence of <i>O</i>-(2-acetamido-2-deoxy-D-glucopyroanosylidene)-amino-<i>N</i>-phenylcarbamate (10 or 100 µM PUGNAC). (D) Graphs showed relative <i>O</i>-GlcNAc (left) and myc-Nkx2.5 (right) levels normalized to the tubulin levels in PUGNAC-treated and untreated cells. The treatment with STZ and PUGNAC significantly increased the <i>O</i>-GlcNAcylation of intracellular proteins, but decreased the myc-Nkx2.5 protein significantly. <i>O</i>-GlcNAcylation levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC-treated myc-Nkx2.5 cells. The myc-Nkx2.5 protein levels were compared in untreated myc-Nkx2.5 cells <i>vs</i> PUGNAC treated myc-Nkx2.5 cells. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05.</p

Expression of Nkx2.5 in the heart tissue of diabetic mice (DM).

<p>(A) Immunoblotting analysis of Nkx2.5 and <i>O</i>-GlycNAcylated proteins in the heart tissue of DM intraperitoneally injected with streptozotocine (180 mg/kg body weight). (B) Graphs showed relative <i>O</i>-GlcNAc (left) and Nkx2.5 (right) levels normalized to the tubulin levels in DM and control. Nkx2.5 protein in diabetic heart of mice was significantly decreased. <i>O</i>-GlcNAcylation and Nkx2.5 protein levels were compared in DM <i>vs</i> control. All values are presented as the mean±standard error of three independent experiments. *<i>p</i><0.05 DM vs. control.</p

Modification of Nkx2.5 by <i>O</i>-GlcNAc.

<p>(A) The lysates of myc-Nkx2.5 and OGT-flag-co-transfected HEK293 cells were subjected to immunoprecipitation with anti-myc antibody and immunoblottings were performed with antibodies against <i>O</i>-GlcNAc (CTD110.6, RL-2) and myc. The modification of myc-Nkx2.5 with O-GlcNAc was detected. (B) Diabetic mice (DM) maintained elevated blood glucose level (>430 mg/dL) at 3 and 7 days after injection with streptozotocine (180 mg/kg body weight). The heart homogenates of control and diabetic mice were immunoprecipitated with anti-Nkx2.5 antibody, followed by immunoblottings with RL-2 antibodies against <i>O</i>-GlcNAc and anti-Nkx2.5 antibody. Nkx2.5 proteins of heart tissues were modified with <i>O</i>-GlcNAc and this modification increased in diabetic mice compared with control mice.</p

Oleic acid (OA) promotes the proliferation and migration ability of MCF10DCIS.COM cells but not SUM225 cells, whereas palmitic acid (PA) leads to cell death in both cells.

<p>(A and B) MTT assay of cell proliferation in MCF10DCIS.COM and SUM225 cells incubated with increasing OA or PA. OA induced significantly increased viability in MCF10DCIS.COM cells but led to cell death in SUM225 cells. PA induced the death of both MCF10DCIS.COM and SUM225 cells. (C) Trans-well assay of cell migration in MCF10DCIS.COM and SUM 225 cells. OA significantly promoted the migration of MCF10DCIS.COM cell but not SUM225 cells. (D) Wound healing assay of lateral migration of MCF10DCIS.COM cells incubated with OA. OA significantly enhanced the lateral migration of MCF10DCIS.COM cells. All the experiments were performed at least in triplicate and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Different Biological Action of Oleic Acid in ALDH^high and ALDH^low Subpopulations Separated from Ductal Carcinoma <i>In Situ</i> of Breast Cancer

<div><p>The mechanisms underlying breast cancer progression of ductal carcinoma in situ <b>(</b>DCIS) associated with fatty acids are largely unknown. In the present study, we compared the action of oleic acid (OA) on two human DCIS cell lines, MCF10DCIS.COM (ER/PR/HER2-negative) and SUM225 (HER2 overexpressed). OA led to a significant increase in proliferation, migration, lipid accumulation and the expression of lipogenic proteins, such as SREBP-1, FAS and ACC-1, in MCF10DCIS.COM cells but not SUM225 cells. The ALDH^high subpopulation analyzed by the ALDEFLUOR assay was approximately 39.2±5.3% of MCF10DCIS.COM cells but was small (3.11±0.9%) in SUM225 cells. We further investigated the different biological action of OA in the distinct ALDH^low and ALDH^high subpopulations of MCF10DCIS.COM cells. OA led to an increase in the expression of ALDH1A1, ALDH1A2 and ALDH1A3 in MCF10DCIS.COM cells. SREBP-1 and ACC-1 were highly expressed in ALDH^high cells relative to ALDH^low cells, whereas FAS was higher in ALDH^low cells. In the presence of OA, ALDH^high cells were more likely to proliferate and migrate and displayed significantly high levels of SREBP-1 and FAS and strong phosphorylation of FAK and AKT relative to ALDH^low cells. This study suggests that OA could be a critical risk factor to promote the proliferation and migration of ALDH^high cells in DCIS, leading to breast cancer progression.</p></div

Oleic acid (OA) promotes the viability and migration through the FAK, PI3K/AKT, and MEK/ERK signaling pathway in MCF10DCIS.COM cells.

<p>(A and B) Representative Western blot and quantitative analysis of phosphorylated FAK, AKT and ERK1/2 in MCF10DCIS.COM cells incubated with OA. OA induced a significant increase in the phosphorylation of FAK, AKT and ERK1/2. (C) MTT assay of cell proliferation in MCF10DCIS.COM cells incubated with OA in the presence of FAK (PF573228), PI3K/AKT (LY294002) and MEK/ERK (PD98059) inhibitors. All kinase inhibitors induced cell death, and OA-promoted proliferation was reduced in the presence of all kinase inhibitors. (D) Trans-well assay of cell migration in MCF10DCIS.COM cells incubated with OA in the presence of FAK, PI3K/AKT and MEK/ERK inhibitors. OA-induced migration was suppressed by the presence of all kinase inhibitors. All the experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p

Oleic acid (OA) further promotes the proliferation and migration abilities and upregulates lipogenic proteins in ALDH^high cells.

<p>(A) Quantitative real-time RT-PCR of ALDH1A1, ALDH1A2 and ALDH1A3 in MCF10DCIS.COM cells. All subtypes of ALDH1 were significantly increased by OA. Notably, OA led to a remarkable increase in ALDH1A2 in MCF10DCIS.COM cells. (B) MTT assay of cell proliferation in ALDH^high and ALDH^low cells incubated with OA. OA-induced proliferation of ALDH^high cells was greater than that of ALDH^low cells. (C) Trans-well assay of cell migration in ALDH^high and ALDH^low cells. The OA-induced migration ability was higher in ALDH^high cells than ALDH^low cells. (D) Representative Western blot of SREBP-1, FAS and ACC-1 in ALDH^high cells and ALDH^low cells. (E, F and G) Analysis of expression levels of SREBP-1, FAS and ACC-1. Significantly higher expression of SERBP-1 and ACC-1 was observed in ALDH^high cells, whereas FAS was significantly higher in ALDH^low cells. OA led to the significant upregulation of SREBP-1 and FAS in ALDH^high cells and the significant upregulation of SREBP-1 and downregulation of FAS in ALDH^low cells. All experiments were performed at least in triplicate, and the values are reported as the means ± standard error. *<i>p</i><0.05, **<i>p</i><0.01.</p