Expression and Clinical Significance of ATP Citrate Lyase in Hepatocellular Carcinoma / 中国医学科学院学报

Objective To investigate the role of ATP citrate lyase(ACLY)in the development of hepatocellular carcinoma(HCC)and the impact of this enzyme on the immune microenvironment of HCC.Methods We utilized the University of Alabama at Birmingham Cancer Data Analysis Portal and the Gene Expression Profiling Interactive Analysis to identify the changes in ACLY expression and prognosis across different tumor types from The Cancer Genome Atlas.With HCC as the disease model,we analyzed the ACLY expression in HCC samples from the gene expression database.Furthermore,we collected the clinical specimens from HCC patients to verify the mRNA and protein levels of ACLY.In addition,we conducted transcriptome sequencing after knocking down the expression of ACLY to analyze the differentially expressed genes and investigated the impact of ACLY expression interference on cell proliferation and other functions.Finally,we explored the correlations of ACLY with immune cells and immune infiltration in the tumor microenvironment,new antigens,and immune checkpoint genes.Results ACLY expression was significantly up-regulated in solid tumors including HCC(all P<0.05),and high ACLY expression was associated with overall survival rate in HCC(P=0.005).Furthermore,high ACLY expression affected the presence of immune cells(e.g.,tumor-associated fibroblasts)and the expression of genes involved in lipid metabolism(all P<0.05).Conclusions ACLY is closely related to the occurrence and development of HCC and lipid metabolism abnormalities.Moreover,it has a specific impact on the immune microenvironment of HCC.

Development of the novel ACLY inhibitor 326E as a promising treatment for hypercholesterolemia

Hepatic cholesterol accumulation is an important contributor to hypercholesterolemia, which results in atherosclerosis and cardiovascular disease (CVD). ATP-citrate lyase (ACLY) is a key lipogenic enzyme that converts cytosolic citrate derived from tricarboxylic acid cycle (TCA cycle) to acetyl-CoA in the cytoplasm. Therefore, ACLY represents a link between mitochondria oxidative phosphorylation and cytosolic de novo lipogenesis. In this study, we developed the small molecule 326E with an enedioic acid structural moiety as a novel ACLY inhibitor, and its CoA-conjugated form 326E-CoA inhibited ACLY activity with an IC50 = 5.31 ± 1.2 μmol/L in vitro. 326E treatment reduced de novo lipogenesis, and increased cholesterol efflux in vitro and in vivo. 326E was rapidly absorbed after oral administration, exhibited a higher blood exposure than that of the approved ACLY inhibitor bempedoic acid (BA) used for hypercholesterolemia. Chronic 326E treatment in hamsters and rhesus monkeys resulted in remarkable improvement of hyperlipidemia. Once daily oral administration of 326E for 24 weeks prevented the occurrence of atherosclerosis in ApoE-/- mice to a greater extent than that of BA treatment. Taken together, our data suggest that inhibition of ACLY by 326E represents a promising strategy for the treatment of hypercholesterolemia.

Metabolic dysregulation and emerging therapeutical targets for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is an aggressive human cancer with increasing incidence worldwide. Multiple efforts have been made to explore pharmaceutical therapies to treat HCC, such as targeted tyrosine kinase inhibitors, immune based therapies and combination of chemotherapy. However, limitations exist in current strategies including chemoresistance for instance. Tumor initiation and progression is driven by reprogramming of metabolism, in particular during HCC development. Recently, metabolic associated fatty liver disease (MAFLD), a reappraisal of new nomenclature for non-alcoholic fatty liver disease (NAFLD), indicates growing appreciation of metabolism in the pathogenesis of liver disease, including HCC, thereby suggesting new strategies by targeting abnormal metabolism for HCC treatment. In this review, we introduce directions by highlighting the metabolic targets in glucose, fatty acid, amino acid and glutamine metabolism, which are suitable for HCC pharmaceutical intervention. We also summarize and discuss current pharmaceutical agents and studies targeting deregulated metabolism during HCC treatment. Furthermore, opportunities and challenges in the discovery and development of HCC therapy targeting metabolism are discussed.

Research progress on ATP citrate lyase (ACLY) inhibitors for decreasing blood lipids / 药学学报

Cardiovascular disease is a principal cause of morbidity and death in the world. Although drug therapy has made great progress in the past few decades, there are still many deficiencies in the prevention and treatment of cardiovascular disease. Dyslipidemia is still a common risk feature and is not sufficiently controlled. A growing body of evidence suggests that the occurrence and development of cardiovascular disease is associated with many associated risk factors, such as higher low-density lipoprotein levels, lower high-density lipoprotein levels and high triglyceride levels. A number of clinical trials in patients with dyslipidemia have shown that actively decreasing low density lipoprotein cholesterol can significantly decrease cardiovascular events. ATP citrate lyase (ACLY) is a cytoplasmic homo-tetrameric enzyme. In the presence of adenosine triphosphate (ATP), ACLY catalyzes the conversion of citric acid and coenzyme A to acetyl-CoA and oxalyl acetate. ACLY is the main enzyme for the production of cytoplasmic acetyl-CoA, and cytoplasmic acetyl-CoA is the precursor required for de novo synthesis of cholesterol and fatty acids. Therefore, it is possible to reduce the production of acetyl-CoA and reduce the levels of cholesterol and triglycerides by inhibiting ACLY. ACLY can be used as a molecular target for reducing blood lipids, and there are an increasing number of studies on ACLY inhibitors. In this paper, the structure and mechanism of ACLY and its relationship with lipid metabolism are briefly introduced, and we review some current ACLY inhibitors.

Effects of downregulated ATP citrate lyase on the growth and apoptosis of prostate cancer cells / 医学研究生学报

Objective Tumor cells are able to support their malignant proliferation by changing metabolic models .Prostate cells rely much on lipid metabolism in which ATP-citrate lyase ( ACLY) plays a very important role .The aim of this research was to study the effects of downregulated ACLY on the cell proliferation , cycle distribution and apoptosis of androgen-independent prostate cancer cells DUl45. Methods DU145 cells were divided into two groups:the cells in experiment group were transfected with the small interfering RNA-mediated knockdown of ACLY , while the cells in control group were transfected with meaningless small interfering RNA.Cell counting Kit test ( CCK-8 ) was applied to detect the effects of the downregulation of ATP citrate lyase on the proliferation of DU145.Flow cytometry instrument was used to analyze the variation of cell cycle distribution and apoptosis rate between groups .Western blot was used to detect the change of intracellular Caspase-3 protein content. Results Western blot showed favorable effects of ACLY interference .Compared with control group , ACLY protein content significantly decreased in experiment group ( P0.05), while the percentage of G2 cells decreased and the percentage of S cells in-creased with most cell cycle blocking at G 0/G1 stage, which were of significant difference .Meanwhile the expression of apoptosis pro-tein Caspase-3 upregulated significantly . Conclusion ACLY is of vital significance to maintain the malignant proliferation of prostate cancer cells and its downregulation results in the inhibition of cell proliferation and the promotion of cell apoptosis .

Nutraceuticals as potential therapeutic agents for colon cancer: a review

Colon cancer is a world-wide health problem and the second-most dangerous type of cancer, affecting both men and women. The modern diet and lifestyles, with high meat consumption and excessive alcohol use, along with limited physical activity has led to an increasing mortality rate for colon cancer worldwide. As a result, there is a need to develop novel and environmentally benign drug therapies for colon cancer. Currently, nutraceuticals play an increasingly important role in the treatment of various chronic diseases such as colon cancer, diabetes and Alzheimer׳s disease. Nutraceuticals are derived from various natural sources such as medicinal plants, marine organisms, vegetables and fruits. Nutraceuticals have shown the potential to reduce the risk of colon cancer and slow its progression. These dietary substances target different molecular aspects of colon cancer development. Accordingly, this review briefly discusses the medicinal importance of nutraceuticals and their ability to reduce the risk of colorectal carcinogenesis.

Characterization of regulatory elements on the promoter region of human ATP-citrate lyase

ATP-citrate lyase (ACL), an enzyme catalyzing the first step in biosynthesis of fatty acids, is induced during the lipogenesis and cholesterologenesis. We demonstrate that the region -213 to -128 of human ACL promoter is responsible for conferring glucose-mediated transcription. This region in the ACL promoter contains Sp1 binding sites determined by DNase I foot-printing assay. Gel retardation assay using oligonucleotides from -179 to -141 and -140 to -110 showed two specific DNA-protein complexes postulated to be formed by transcription factor Sp1. Competition gel shift and supershift assays have confirmed that these DNA-protein complexes were the result of induced Sp1 as well as another Sp1-related proteins. Western blot analysis also demonstrated that transcription factor Sp1 was slightly increased in the nuclear proteins extracted from Alexander cells following supplementation of glucose. In addition, expression of 110 kDa protein reacting with antibody against Sp3 was dramatically increased by glucose supplementation, while isoforms of Sp3, about 80 kDa in size was decreased in its amounts. Our results suggest that changes in the expression of Sp1 family proteins play an important role in activation of the ACL promoter by glucose.

Regulation of ATP-citrate lyase gene transcription

It has been suggested that glucose metabolites and insulin are the most important factors inducing ATP-citrate lyase (ACL) by a high carbohydrate diet. We have used a primary culture of rat hepatocytes to confirm the role of glucose and insulin in terms of ACL gene expression. The results showed that glucose displayed a direct effect on ACL gene expression and the insulin helps the glucose effect. The nucleotide sequences from -512 to -485 of the ACL promoter are highly homologous (70%) to the sequences surrounding the carbohydrate response element (ChoRE) of the S14 gene. The gel retardation analysis using ChoRE of the S14 gene showed that the ACL promoter which contains the ChoRE-like sequence specifically inhibited the formation of the complex by the nuclear proteins isolated from rat liver. To localize the regions which are involved in the regulation of ACL gene expression, transient expression assay using ACL promoter-CAT (chloramphenicol acetyltransferase) constructs containing various lengths of a 5' flanking region of the ACL gene were carried out. The proximal promoter region -419 to -1 containing several potential Sp1 binding sites showed the strong enhancing effect, which increases the transcription of CAT genes in the various cell lines, such as the CHO (Chinese hamster ovary) cell, the HepG2 cell, and primary cultured rat hepatocytes. In response to glucose, among the ACL promoter-CAT constructs, only pNP33-CAT (-1342 to -1) showed a 2.64 fold increase in CAT activity by a high concentration of glucose. The activation of ACL gene expression by glucose seems to be regulated in a complicated manner involving interactions between the contexts of the several sequence elements and various transacting factors, which is not a simple mechanism directed only by a short sequence element.

Induction of hepatic ATP-citrate lyase by insulin in diabetic rat--effects of insulin on the contents of enzyme and its mRNA in cytosol, and the transcriptional activity in nuclei

The effects of insulin on ATP-citrate lyase, its mRNA in cytosol, and the transcriptional activity in nuclei of diabetic rat liver were studied. Experimental diabetes was induced by an intraperitoneal injection of streptozotocin, and livers were removed from rats at 0, 1, 3, 6, 16, and 72 hours after the administration of insulin. ATP-citrate lyase began to increase at 16 hours, and continuously increased until 72 hours. The amount of mRNA encoding ATP-citrate lyase increased abruptly at 16 hours, then decreased to near basal level in 72 hours. No change in the transcription rate was observed until 3 hours after insulin administration. However, the activity increased 4-fold at 6 hours and 7-fold at 16 hours, 16-fold at 6 hours and 28-fold at 16 hours when pGACL1 and pGACL2 were used as probes, respectively, preceding the increase in the amounts of mRNA and the enzyme. It is suggested that the increase in the amount of ATP-citrate lyase by insulin is primarily due to the increase in the transcriptional activity of the gene in nuclei, which results in the subsequent increase in the amount of mRNA for the biosynthesis of ATP-citrate lyase in cytosol.

Relationship between fatty acid binding proteins, acetyl-CoA formation and fatty acid synthesis in developing human placenta.

The relationship between fatty acid binding proteins, ATP citrate lyase activity and fatty acid synthesis in developing human placenta has been studied. Fatty acid binding proteins reverse the inhibitory efect of palmitoyl-CoA and oleate on ATP citrate lyase and fatty acid synthesis. In the absence of these inhibitors fatty acid binding proteins activate ATP citrate lyase and stimulate [ 1–14 C] acetate incorporation into placental fatty acids indicating binding of endogenous inhibitors by these proteins. Thus these proteins regulate the supply of acetyl-CoA as well as the synthesis of fatty acids from that substrates. As gestation proceeds and more lipids are required by the developing placenta fatty acid binding protein content, activity of ATP citrate lyase and rate of fatty acid synthesis increase indicating a cause and efect relationship between the demand of lipids and supply of precursor fatty acids during human placental development.