ATP citrate lyase (ACL or ACLY) is an extra-mitochondrial enzyme widely distributed in various human and animal tissues. ACL links glucose and lipid metabolism by catalyzing the formation of acetyl-CoA and oxaloacetate from citrate produced by glycolysis in the presence of ATP and CoA. ACL is aberrantly expressed in many immortalized cells and tumors, such as breast, liver, colon, lung and prostate cancers, and is correlated reversely with tumor stage and differentiation, serving as a negative prognostic marker. ACL is an upstream enzyme of the long chain fatty acid synthesis, providing acetyl-CoA as an essential component of the fatty acid synthesis. Therefore, ACL is a key enzyme of cellular lipogenesis and potent target for cancer therapy. As a hypolipidemic strategy of metabolic syndrome and cancer treatment, many small chemicals targeting ACL have been designed and developed. This review article provides an update for the research and development of ACL inhibitors with a focus on their patent status, offering a new insight into their potential application.
The aim of this study is to construct a lentiviral expression vector containing a scavenger receptor (SR-PSOX) that binds with uniquely phosphatidylserine and oxidized lipoprotein with six histidine tags and to investigate the function of SR-PSOX in atherosclerosis. We utilize the ViraPower lentiviral expression system which was efficient to deliver in vitro or in vivo the target gene into dividing and non-dividing mammalian cells using an enhanced biosafety replication-incompetent lentivirus. The blunt-end sequence was amplified using the reverse transcription-polymerase chain reaction and directional TOPO cloning reaction. Through a pair of the cytomegalovirus forward primer and the reverse primer of SR-PSOX, the correct clones were identified by polymerase chain reaction and sequencing. The ViraPower packaging mix and SR-PSOX-pLenti6/V5 TOPO expression plasmid were co-transfected into the 293FT cell line using Lipofectamine 2000. The expression of endogenous and exogenous SR-PSOX as well as tumor necrosis factor (TNF)-alpha protein in various foam cell models at different time points were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot and indirect immunofluorescence assay. Western blot and immunofluorescence analysis confirmed that the expressions of SR-PSOX and TNF-alpha protein were upregulated in foam cell models. Our data suggested that the overexpression of recombinant human SR-PSOX protein can promote foam cell formation and upregulate the expression of the inflammatory factor TNF-alpha.
Using human vascular endothelial cells (ECV304) as the target, we studied the effect of caveolin (CAV)-1 in the course of insulin-stimulated expression of plasminogen activator inhibitor (PAI)-1. The appropriate single-stranded oligonucleotides representing the RNAi CAV-1 gene were analyzed by Ambion software. After annealing to generate double-stranded oligonucleotides (ds oligo), it was cloned into the pENTR/U6 entry vector containing RNA polymerase III expression element by T4 DNA ligase. The short hairpin (shRNA) sequences transferred from the pENTR/U6 entry were cloned into the pLenti6/BLOCK-iT-DEST vector with an LR recombination reaction. After identification by sequencing, we successfully constructed the CAV-1 RNAi lentiviral expression system using Gateway technology. Silencing efficiency was assayed by real-time reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blotting. ECV304 cells were cultured in the medium containing different concentrations of insulin (1x10(-9) to 1x10(-7) M) with the CAV-1 gene silenced or not. The expression level and subcellular localization of PAI-1 and CAV-1 were compared using reverse transcription-polymerase chain reaction, immunofluorescence staining and Western blot assay. The results showed that the potent inhibition of CAV-1 expression could reach 85%, and it was specific to the CAV-1-derived shRNA, not the S100A13-derived shRNA. There was no dramatic difference in PAI-1 expression between the RNAi+ and RNAi- ECV304 cells incubated with physiological insulin, but PAI-1 protein did accumulate under the cell membrane. As the concentration of insulin increased, the expression of PAI-1 was up-regulated, whereas the expression of CAV-1 attenuated. Furthermore, PAI-1 clearly augmented after CAV-1 knockdown. These results indicated that hyperinsulinism could promote PAI-1 expression by inhibiting CAV-1, and stabilizing or up-regulating CAV-1 expression in endothelial cells might reduce complications of the great vessels and capillary vessels in diabetes.
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