Supplementary Materialsijms-20-06268-s001. reactive air species formation set alongside the p53+/+ cell series. The reactive air types (ROS) scavengers N-acetyl cysteine (NAC) and decreased glutathione (GSH) partly attenuated apoptosis in the HCT116 p53?/? cell series but acquired no obvious influence on the p53+/+ cell series. Furthermore, p53 induced the appearance of its downstream focus on genes, and and were significantly induced by palmitic acid. Loss of p21 prospects to more cellular apoptosis compared with wide type in the HCT116 cell collection under PA stress. To gain insight into palmitic acids physiological effects, we constructed an animal model of obesity by feeding a high fat diet comprising PA to p53+/+ and p53?/? mice. Among mice receiving the high fat diet, glucose tolerance test assays showed higher glucose levels in p53?/? mice than crazy type mice; there were no variations between p53+/+ and p53?/? mice that received the regular diet. In summary, p53 may protect cells against lipotoxicity through downstream target genes by eliminating palmitic acid-induced ROS production. 2. Results 2.1. Palmitic Acid Activates p53 inside a Dose-and Time-Dependent Manner Palmitic acid is definitely a saturated free fatty acid that builds up in non-adipose cells, leading to cell liopotoxicity effects such as apoptosis and disease onset [30]. For example, palmitic acid can induce pancreatic -cell dysfunction, resulting in insulin resistance and diabetes [6,30,31], and cardiac myocyte apoptosis, leading to heart failure [13,32]. p53 is definitely a tumor suppressor, Walrycin B which is definitely often viewed as a cellular guardian that protects cells from damage [33,34]. In order to investigate p53s part in palmitic acid-induced lipotoxicity, a complex of palmitic acid and albumin with defined percentage was used to mimic saturated free fatty acids under physiological conditions. Although the normal physiological percentage of fatty acid to albumin is about 2:1, a percentage of 8:1 palmitic acid:bovine serum albumin (BSA) complex (hereafter abbreviated as PA) was used in this experimental system [1]. HCT116 p53+/+ and HCT116 p53?/? cell lines were separately complemented with 0, 50, 100, or 250 M PA or BSA for 24 h. p53 was mildly triggered by PA Walrycin B in the HCT116 p53+/+ cell collection inside a dose- and time-dependent manner (Number 1A,C). Manifestation levels of the important p53 target gene, (cyclin-dependent kinase inhibitor 1), also increased significantly after PA treatment (Number 1A,C). Levels of p21 protein improved by approximately 2-fold relative to the BSA control in HCT116 p53+/+ cells (Number 1B,D). Interestingly, oleic acid complexed with BSA in an 8:1 percentage (hereafter abbreviated as OA) did not activate p53 as PA did (Number 1E,F). These results demonstrate that p53 was specifically induced by PA. Palmitic acid activates p53 inside a dose- and time-dependent manner and may play an important part in lipotoxicity induced by PA. Open in a separate window Number 1 Palmitic acid induces p53 manifestation in human colon carcinoma cells (HCT116) inside a dose- and time-dependent manner. (A) HCT116 p53+/+ and p53?/? cell lines were treated with the indicated dose of palmitic acid. A total of 40 g of total protein draw out was resolved on SDS-PAGE. Immunoblotting was performed using p53 and p21 antibodies, and -actin was used as a loading control. (B) Quantitative analysis of (A) by ImageJ software. (C) HCT116 cells were treated with 250 M palmitic acid at indicated time points; 40 g of total protein draw out was resolved on SDS-PAGE. Immunoblotting was performed using p53 and p21 antibodies, and -actin was used as a loading control. (D) Quantitative analysis of (C) by ImageJ software. (E) Palmitic acid, not oleic acid, specifically activated p53 expression. HCT116 cells were treated with 250 M oleic acid and 250 M palmitic acid for 24 h. Immunoblotting was performed using p53 and p21 antibodies. (F) Quantitative analysis of E by ImageJ software. The data are indicated as mean SE of three self-employed experiments. 2.2. Cells are more Sensitive to PA in HCT116 p53?/? Cell Collection Compared to HCT116 p53+/+ Cell Collection To further investigate p53s part in PA treatment, HCT116 p53+/+ and HCT116 p53?/? cells were treated with different PA doses and incubated with PA for numerous time periods. First, we observed that HCT116 p53?/? cells became rounder and gradually broke away from the plate faster than HCT116 p53+/+ cells. Walrycin B To further observe the long-term effects of PA treatment, we seeded HCT116 p53+/+ and p53?/? cells into Rabbit polyclonal to ACAD9 6-well plates at (4 104) 5%.