Comparative Proteomic Analysis Reveals the Upregulation of Ketogenesis in Cardiomyocytes Differentiated from Induced Pluripotent Stem Cells.

Diverse metabolic pathways, such as the tricarboxylic acid cycle, pyruvate metabolism, and oxidative phosphorylation, regulate the differentiation of induced pluripotent stem cells (iPSCs) to cells of specific lineages and organs. Here, the protein dynamics during cardiac differentiation of human iPSCs into cardiomyocytes (CMs) are characterized. The differentiation is induced by N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide, a Wnt signaling inhibitor, and confirmed by the mRNA and protein expression of cTnT and MLC2A in CMs. For comparative proteomics, cells from three stages, namely, hiPSCs, cardiac progenitor cells, and CMs, are prepared using the three-plex tandem mass tag labeling approach. In total, 3970 proteins in triplicate analysis are identified. As the result, the upregulation of proteins associated with branched chain amino acid degradation and ketogenesis by the Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis are observed. The levels of 3-hydroxymethyl-3-methylglutaryl-CoA lyase, 3-hydroxymethyl-3-methylglutaryl-CoA synthase 2, and 3-hydroxybutyrate dehydrogenase 1, involved in ketone body metabolism, are determined using western blotting, and the level of acetoacetate, the final product of ketogenesis, is higher in CMs. Taken together, these observations indicate that proteins required for the production of diverse energy sources are naturally self-expressed during cardiomyogenic differentiation. Furthermore, acetoacetate concentration might act as a regulator of this differentiation.

Quantitative Proteomic Analysis of Changes Related to Age and Calorie Restriction in Rat Liver Tissue.

Calorie restriction (CR) is the most frequently studied mechanism for increasing longevity, protecting against stress, and delaying age-associated diseases. Most studies have initiated CR in young animals to determine the protective effects against aging. Although aging phenomena are well-documented, the molecular mechanisms of aging and CR remain unclear. In this study, we observe changes in hepatic proteins upon age-related and diet-restricted changes in the rat liver using quantitative proteomics. Quantitative proteomes are measured using tandem mass tag labeling followed by LC-MS/MS. We compare protein levels in livers from young (6 months old) and old (25 months old) rats with 40% calorie-restricted (YCR and OCR, respectively) or ad libitum diets. In total, 44 279 peptides and 3134 proteins are identified and 260 differentially expressed proteins are found. Functional enrichment analysis show that these proteins are mainly involved in glucose and fatty acid metabolism-related processes, consistent with the theory that energy metabolism regulation is dependent on age-related and calorie-restricted changes in liver tissue. In addition, proteins mediating inflammation and gluconeogenesis are increased in OCR livers, but not YCR livers. These results show that CR in old rats might not have antiaging benefits because liver inflammation is increased.

LDHA Desuccinylase Sirtuin 5 as A Novel Cancer Metastatic Stimulator in Aggressive Prostate Cancer.

Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men worldwide. Around 80% of the patients who developed advanced PCa suffered from bone metastasis, with a sharp drop in the survival rate. Despite great efforts, the detailed mechanisms underlying castration-resistant PCa (CRPC) remain unclear. Sirtuin 5 (SIRT5), an NAD+-dependent desuccinylase, is hypothesized to be a key regulator of various cancers. However, compared to other SIRTs, the role of SIRT5 in cancer has not been extensively studied. Here, we revealed significantly decreased SIRT5 levels in aggressive PCa cells relative to the PCa stages. The correlation between the decrease in the SIRT5 level and the patient's reduced survival rate was also confirmed. Using quantitative global succinylome analysis, we characterized a significant increase in the succinylation at lysine 118 (K118su) of lactate dehydrogenase A (LDHA), which plays a role in increasing LDH activity. As a substrate of SIRT5, LDHA-K118su significantly increased the migration and invasion of PCa cells and LDH activity in PCa patients. This study reveals the reduction of SIRT5 protein expression and LDHA-K118su as a novel mechanism involved in PCa progression, which could serve as a new target to prevent CPRC progression for PCa treatment.

SIRT5 Directly Inhibits the PI3K/AKT Pathway in Prostate Cancer Cell Lines.

BACKGROUND/AIM: Prostate cancer (PCa) is the most commonly diagnosed genital cancer in men globally. Among patients who develop advanced PCa, 80% are affected by bone metastasis, with a sharp drop in survival rate. Despite efforts, the details of mechanisms of metastasis of PCa remain unclear. SIRT5, an NAD+-dependent deacylase, is hypothesized to be a crucial regulator of various cancers. The role of SIRT5 in cancer has not been extensively studied compared to other SIRTs. In this study, we showed significantly decreased levels of SIRT5 in PC-3M, a highly aggressive PC-3 cell variant. MATERIALS AND METHODS: We characterized the differentially expressed proteins between parental and SIRT5 KO PC-3 cells using quantitative proteomics analysis. RESULTS: A significant increase in expression of interleukin-1ß (IL-1ß) in SIRT5 KO cells was observed, and the PI3K/AKT/NF-ĸB signaling pathway was found significantly elevated in SIRT5 KO cells by the Gene Ontology annotation and KEGG pathway functional enrichment analysis. Moreover, we confirmed that SIRT5 can bind PI3K by immunoprecipitation analysis. CONCLUSION: This study is the first to demonstrate a relationship between SIRT5 and PCa metastasis, suggesting that SIRT5-mediated inhibition of the PI3K/AKT/NK-kB pathway is reduced for secondary metastasis from bone to other tissues.

Correction: Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

[This corrects the article DOI: 10.1371/journal.pone.0220807.].

Secretome profiling of PC3/nKR cells, a novel highly migrating prostate cancer subline derived from PC3 cells.

Prostate cancer (PCa) is the most common cancer among men worldwide. Most PCa cases are not fatal; however, the outlook is poor when PCa spreads to another organ. Bone is the target organ in about 80% of patients who experience metastasis from a primary PCa tumor. In the present study, we characterized the secretome of PC3/nKR cells, which are a new subline of PC3 cells that were originally isolated from nude mice that were implanted with PC3 cells without anti-natural killer (NK) cell treatment. Wound healing and Transwell assays revealed that PC3/nKR cells had increased migratory and invasive activities in addition to a higher resistance to NK cells-induced cytotoxicity as compared to PC3 cells. We quantitatively profiled the secreted proteins of PC3/nKR and PC3 cells by liquid chromatography-tandem mass spectrometry analysis coupled with 2-plex tandem mass tag labeling. In total, 598 secretory proteins were identified, and 561 proteins were quantified, among which 45 proteins were secreted more and 40 proteins were secreted less by PC3/nKR cells than by PC3 cells. For validation, the adapter molecule crk, serpin B3, and cystatin-M were analyzed by western blotting. PC3/nKR cells showed the selective secretion of NKG2D ligand 2, HLA-A, and IL-6, which may contribute to their NK cell-mediated cytotoxicity resistance, and had a high secretion of crk protein, which may contribute to their high migration and invasion properties. Based on our secretome analysis, we propose that PC3/nKR cells represent a new cell system for studying the metastasis and progression of PCa.

Comparative Secretome Profiling and Mutant Protein Identification in Metastatic Prostate Cancer Cells by Quantitative Mass Spectrometry-based Proteomics.

BACKGROUND: Secreted proteins play an important role in promoting cancer (PCa) cell migration and invasion. Proteogenomics helps elucidate the mechanism of diseases, discover therapeutic targets, and generate biomarkers for diagnosis through protein variations. MATERIALS AND METHODS: We carried out mass a spectrometry-based proteomic analysis of the conditioned media (CM) from two human prostate cancer cell lines, belonging to different metastatic sites, to identify potential metastatic and/or aggressive factors. RESULTS: We identified a total of 598 proteins, among which 561 were quantified based on proteomic analysis. Among the quantified proteins, 128 were up-regulated and 83 were down-regulated in DU145/PC3 cells. Six mutant peptides were identified in the CM of prostate cancer cell lines using proteogenomics approach. CONCLUSION: This is the first proteogenomics study in PCa aiming at exploring a new type of metastatic factor, which are mutant peptides, predicting a novel biomarker of metastatic PCa for diagnosis, prognosis and drug targeting.

Protective Effect of Isoliquiritigenin against Ethanol-Induced Hepatic Steatosis by Regulating the SIRT1-AMPK Pathway.

Ethanol-induced fat accumulation, the earliest and most common response of the liver to ethanol exposure, may be involved in the pathogenesis of liver diseases. Isoliquiritigenin (ISL), an important constituent of Glycyrrhizae Radix, is a chalcone derivative that exhibits antioxidant, anti-inflammatory, and phytoestrogenic activities. However, the effect of ISL treatment on lipid accumulation in hepatocytes and alcoholic hepatitis remains unclear. Therefore, we evaluated the effect and underlying mechanism of ISL on ethanol-induced hepatic steatosis by treating AML-12 cells with 200 mM ethanol and/or ISL (0~50 µM) for 72 hr. Lipid accumulation was assayed by oil red O staining, and the expression of sirtuin1 (SIRT1), sterol regulatory element-binding protein-1c (SREBP-1c), AMP-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor alpha (PPARα) was studied by western blotting. Our results indicated that ISL treatment upregulated SIRT1 expression and downregulated SREBP-1c expression in ethanol-treated cells. Similarly, oil red O staining revealed a decrease in ethanol-induced fat accumulation upon co-treatment of ethanol-treated cells with 10, 20, and 50 µM of ISL. These findings suggest that ISL can reduce ethanol induced-hepatic lipogenesis by activating the SIRT1-AMPK pathway and thus improve lipid metabolism in alcoholic fatty livers.