Metabolic switches during the first steps of adipogenic stem cells differentiation

AbstractThe understanding of metabolism during cell proliferation and commitment provides a greater insight into the basic biology of cells, allowing future applications. Here we evaluated the energy and oxidative changes during the early adipogenic differentiation of human adipose tissue-derived stromal cells (hASCs). hASCs were maintained under differentiation conditions during 3 and 7days. Oxygen consumption, mitochondrial mass and membrane potential, reactive oxygen species (ROS) generation, superoxide dismutase (SOD) and catalase activities, non-protein thiols (NPT) concentration and lipid peroxidation were analyzed. We observed that 7days of adipogenic induction are required to stimulate cells to consume more oxygen and increase mitochondrial activity, indicating organelle maturation and a transition from glycolytic to oxidative energy metabolism. ROS production was only increased after 3days and may be involved in the differentiation commitment. ROS source was not only the mitochondria and we suggest that NOX proteins are related to ROS generation and therefore adipogenic commitment. ROS production did not change after 7days, but an increased activity of catalase and NPT concentration as well as a decreased lipid peroxidation were observed. Thus, a short period of differentiation induction is able to change the energetic and oxidative metabolic profile of hASCs and stimulate cytoprotection processes

Selective Cytotoxicity of 1,3,4-Thiadiazolium Mesoionic Derivatives on Hepatocarcinoma Cells (HepG2).

In this work, we evaluated the cytotoxicity of mesoionic 4-phenyl-5-(2-Y, 4-X or 4-X-cinnamoyl)-1,3,4-thiadiazolium-2-phenylamine chloride derivatives (MI-J: X=OH, Y=H; MI-D: X=NO2, Y=H; MI-4F: X=F, Y=H; MI-2,4diF: X=Y=F) on human hepatocellular carcinoma (HepG2), and non-tumor cells (rat hepatocytes) for comparison. MI-J, M-4F and MI-2,4diF reduced HepG2 viability by ~ 50% at 25 μM after 24-h treatment, whereas MI-D required a 50 μM concentration, as shown by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The cytotoxicity was confirmed with lactate dehydrogenase assay, of which activity was increased by 55, 24 and 16% for MI-J, MI-4F and MI-2,4diF respectively (at 25 μM after 24 h). To identify the death pathway related to cytotoxicity, the HepG2 cells treated by mesoionic compounds were labeled with both annexin V and PI, and analyzed by flow cytometry. All compounds increased the number of doubly-stained cells at 25 μM after 24 h: by 76% for MI-J, 25% for MI-4F and MI-2,4diF, and 11% for MI-D. It was also verified that increased DNA fragmentation occurred upon MI-J, MI-4F and MI-2,4diF treatments (by 12%, 9% and 8%, respectively, at 25 μM after 24 h). These compounds were only weakly, or not at all, transported by the main multidrug transporters, P-glycoprotein, ABCG2 and MRP1, and were able to slightly inhibit their drug-transport activity. It may be concluded that 1,3,4-thiadiazolium compounds, especially the hydroxy derivative MI-J, constitute promising candidates for future investigations on in-vivo treatment of hepatocellular carcinoma

Annexin V-FITC and propidium iodide staining of HepG2 treated with 1,3,4-thiadiazolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 μM for 18–24 h. Then, the cells were collected with trypsin and 10.000 events were analyzed by flow cytometry by FL2 and FL1 filters. (A) control, (B) MI-D, (C) MI-J, (D) MI-4F and (E) MI-2,4diF. The figures show representative dot-plot with the different cell populations: left-bottom = labeled cells; left-top = PI labeled; right-top = doubly labeled; right-bottom = annexin V labeled. The results were expressed as mean ± SD of three independents experiments.</p

Effects of 1,3,4-thiadiazolium derivatives on hepatocytes morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>Hepatocytes were incubated with the derivatives at 25 μM for 24 h. The images were obtained using inverted microscope. A: control (untreated cells); B-E: treatments by MI-D, MI-J, MI-4F and MI-2,4diF, respectively. The scale is indicated by black bars representing 50 μm. The photographs represent three different experiments in triplicate.</p

Annexin V-FITC and propidium iodide staining of hepatocytes treated by 1,3,4-thiadizolium derivatives (the experimental conditions are described in the Materials and Methods section 2.5.3).

<p>Hepatocytes were incubated with derivatives at 25 μM for 20 h. The images (10X magnification) were captured with an AXIOVERT 40CSFL fluorescence microscope. The scale is indicated by white bars representing 100 μm. The annexin V-FITC-positive cells are stained in green, and the PI-positive cells in red. The images represent (A) control (untreated cells), (B) ASA positive control at 20 mM, (C) MI-D, (D) MI-J, (E) MI-4F and (F) MI-2,4diF. The figures represent three different experiments in triplicate.</p

Cytotoxic effects of 1,3,4-thiadiazolium derivatives on HepG2 cells.

<p>A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1). The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5, 25 or 50 μM for 24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions, as described above, LDH activity was measured in supernatants. Data represent means of four different experiments in quadruplicate The results were expressed as % of viability in comparison to control. * and *** denote values significantly different from the control or between the different treatments at <i>P</i>< 0.05 and <i>P</i>< 0.0001, respectively.</p

Effects of 1,3,4-thiadiazolium derivatives on HepG2 cell morphology (the experimental conditions are described in the Materials and Methods section 2.6).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 5 μM for 3 h. The images were captured with a 100X magnification; they correspond to control (A), MI-D (B), MI-J (C), MI-4F (D) and MI-2,4diF (E). The scale is indicated by black bars representing 0.02 mm. The arrows show morphological modifications as blebs, increased volume and vacuolization. The photographs represent three different experiments in triplicate.</p

DNA fragmentation in HepG2 cells, as induced by 1,3,4-thiadiazolium derivatives (the experimental conditions are described in the Materials and Methods section 2.7).

<p>The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 μM for 24 h. For each sample, 10.000 events were analyzed by flow cytometry using FL2 filter. (A) control, (B) MI-D, (C) MI-J, (D) MI-4F and (E) MI-2,4diF. The histograms represent three different experiments in triplicate.</p

Cytotoxic effects of 1,3,4-thiadiazolium derivatives on hepatocytes.

<p>A. MTT assay (the experimental conditions are described in the Materials and Methods section 2.5.1) The cells were seeded with or without 1,3,4-thiadiazolium derivatives at 25 for 18–24 h. The results were expressed as % of viability in comparison to control. B. LDH release assay (the experimental conditions are described in the Materials and Methods section 2.5.2). Under the same treatment conditions described above, LDH activity was measured in the supernatants. Data represent means of four different experiments in quadruplicate. The results were expressed as % of viability in comparison to control. ** and *** denotes values significantly different from the control or between the different treatments at <i>P</i>< 0.01 and <i>P</i>< 0.0001, respectively.</p