Differential growth inhibition, cell cycle arrest and apoptosis of MCF-7 and MDA-MB-231 cells to holocarboxylase synthetase suppression.

Holocarboxylase synthetase (HLCS) catalyzes the covalent attachment of biotin onto the biotin-dependent carboxylases. Recent studies have shown that HLCS is over-expressed in breast cancer patients. Here we investigated the functional roles of free biotin and HLCS in supporting growth and migration of breast cancer cell lines. Depletion of biotin from culture medium markedly reduced biotinylation of the two most abundant biotin-carboxylases, acetyl-CoA carboxylase and pyruvate carboxylase. This was accompanied by a marked decrease in cell growth. Suppression of HLCS expression in the low invasive breast cancer cell line MCF-7 resulted in an 80% reduction of biotinylated ACC, but not PC. HLCS knockdown MCF-7 cell lines showed 40-50% reduction of proliferation and 35% reduction of migration, accompanied by G1 cell cycle-arrest-induced apoptosis. In contrast, knockdown of HLCS expression in the highly invasive cell line MDA-MB-231 resulted in only marginal reduction of biotinylation of both ACC and PC, accompanied by 30% reduction of proliferation and 30% reduction of migration. Our studies provide new insights to use HLCS as a novel anti-cancer drug target.

Pyruvate carboxylase supports basal ATP-linked respiration in human pluripotent stem cell-derived brown adipocytes.

Brown adipocytes (BA) are a specialized fat cell which possesses a high capacity for fuel oxidation combined with heat production. The maintenance of high metabolic activity in BA requires elevated oxidation of fuel through the tricarboxylic acid cycle. Pyruvate carboxylase (PC) was previously proposed to be essential for coordination between fuel oxidation and thermogenesis. By differentiating human pluripotent stem cells to mature BA in vitro, we showed that ablation of PC gene by CRISPR Cas9 genome engineering did not impair the ability of stem cells to generate mature BA. However, brown adipocytes deficient for PC expression displayed a 35% reduction in ATP-linked respiration, but not thermogenesis under both basal and isoproterenol-stimulated conditions. This relatively mild impairment of ATP-link respiration in PC knockout BA was protected by increased spare mitochondrial respiratory capacity. Taken together, this study highlights the role of PC in supporting fuel oxidation rather than thermogenesis in human BA.

The role of the phosphate groups of trinitrophenyl adenosine 5'-triphosphate (TNP-ATP) in allosteric activation of pyruvate carboxylase and the inhibition of acetyl CoA-dependent activation.

A previous study showed that 2'-3'-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate (TNP-ATP) was a weak allosteric activator of Rhizobium etli pyruvate carboxylase (RePC) in the absence of acetyl-CoA. On the other hand, TNP-ATP inhibited the allosteric activation of RePC by acetyl-CoA. Here, we aimed to study the role of triphosphate group of TNP-ATP on its allosteric activation of the enzyme and inhibition of acetyl-CoA-dependent activation of RePC using TNP-ATP and its derivatives, including TNP-ADP, TNP-AMP and TNP-adenosine. The pyruvate carboxylation activity was assayed to determine the effect of reducing the number of phosphate groups in TNP-ATP derivatives on allosteric activation and inhibition of acetyl-CoA activation of RePC and chicken liver pyruvate carboxylase (CLPC). Reducing the number of phosphate groups in TNP-ATP derivatives decreased the activation efficacy for both RePC and CLPC compared to TNP-ATP. The apparent binding affinity and inhibition of activation of the enzymes by acetyl-CoA were also diminished when the number of phosphate groups in the TNP-ATP derivatives was reduced. Whilst TNP-AMP activated RePC, it did not activate CLPC, but it did inhibit acetyl-CoA activation of both RePC and CLPC. Similarly, TNP-adenosine did not activate RePC; however, it did inhibit acetyl-CoA activation using a different mechanism compared to phosphorylated TNP-derivatives. These findings indicate that mechanisms of PC activation and inhibition of acetyl-CoA activation by TNP-ATP and its derivatives are different. This study provides the basis for possible drug development for treatment of metabolic diseases and cancers with aberrant expression of PC.

Targeting Cancer Metabolism and Current Anti-Cancer Drugs.

Several studies have exploited the metabolic hallmarks that distinguish between normal and cancer cells, aiming at identifying specific targets of anti-cancer drugs. It has become apparent that metabolic flexibility allows cancer cells to survive during high anabolic demand or the depletion of nutrients and oxygen. Cancers can reprogram their metabolism to the microenvironments by increasing aerobic glycolysis to maximize ATP production, increasing glutaminolysis and anabolic pathways to support bioenergetic and biosynthetic demand during rapid proliferation. The increased key regulatory enzymes that support the relevant pathways allow us to design small molecules which can specifically block activities of these enzymes, preventing growth and metastasis of tumors. In this review, we discuss metabolic adaptation in cancers and highlight the crucial metabolic enzymes involved, specifically those involved in aerobic glycolysis, glutaminolysis, de novo fatty acid synthesis, and bioenergetic pathways. Furthermore, we also review the success and the pitfalls of the current anti-cancer drugs which have been applied in pre-clinical and clinical studies.

Characterization of intra- and inter-species hybrid tetramers of pyruvate carboxylase: Biotin and the BCCP domain play a crucial role in determination of the kinetics and thermodynamics of catalysis.

The formation, kinetics and thermodynamic activation parameters of hybrid tetramers of pyruvate carboxylase (PC) formed between wild-type Rhizobium etli pyruvate carboxylase (WTRePC) and mutant forms of this enzyme, as well as between Aspergillus nidulans PC and mutant forms of RePC have been characterized in a previous study. In this current work, we aim to extend the previous study by forming hybrid tetramers between WTRePC or chicken liver PC (CLPC) with single or double mutant RePCs. By forming hybrid tetramers between WTRePC with either K1119A or ΔBCCP RePC, the biotin moiety and BCCP (biotin carboxyl carrier protein) domain appear to play a crucial role in determination of thermodynamic activation parameters, especially the activation entropy, and the order of tetrameric structure. Using E218A:K1119A hybrid tetramers, an alternative pathway of biotin carboxylation occurred only in the absence of acetyl CoA. In this pathway, the biotin of the E218A subunits is carboxylated in the BC domain of the K1119A subunits, since the E218A mutation destroys the catalytic activity of the BC domain. Transfer of the carboxyl group to pyruvate could then occur in the CT domain of either E218A or K1119A. Part of the reduction of activity in hybrid tetramers of WTRePC and double mutant, E218A.K1119A could result from the loss of this pathway. Previously, D1018A mutant RePC homotetramers exhibited a 12-fold increase in the rate constant for catalysis in the absence of acetyl CoA. This was taken to indicate that inter-residue interactions involving D1018 inhibit the interconversion between the symmetrical and asymmetrical forms of the tetramer in the absence of acetyl CoA. The mutation, D1018A, in hybrid tetramers of WTRePC:D1018A.K1119A (D1018A.K1119A is a double mutant form of RePC) had no such effect on the rate constant, suggesting that in hybrid tetramers obligatory oscillation between asymmetrical and symmetrical conformers of the tetramer is not required to drive the catalytic cycle. Finally, K1119A or E218A RePC mutant can form hybrid tetramers with PC subunits from an evolutionarily distant species, chicken, that have stability characteristics that lie between those of the homotetramers of the two enzymes. This work provides insights into the how the PC tetramer functions to perform catalysis and is regulated by acetyl CoA. The ability to form hybrid tetrameric PCs composed of PC subunits from widely varying species that have a mixture of characteristics of the two source enzymes may also provide ways of developing novel PCs for biotechnological purposes.

Characterization of the kinetics and activation thermodynamics of intra- and inter-organism hybrid tetramers of pyruvate carboxylase.

In sedimentation velocity experiments, we have been able to detect hybrid Rhizobium etli pyruvate carboxylase tetramers formed between subunits that contain covalently bound biotin and mutant subunits that do not. This was performed by forming complexes of the tetramers with the biotin-binding protein avidin. In addition, we have shown that it is possible to form hybrid tetramers of pyruvate carboxylase subunits from two different organisms (bacteria - Rhizobium etli and fungi - Aspergillus nidulans). In hybrid tetramers containing mutant subunits that are not fully catalytically active and fully catalytically active subunits, the catalytic and regulatory properties of these hybrid tetramers are modified compared to homotetramers of the fully active pyruvate carboxylase subunits. Our data indicates that the model of catalysis involving half-of-the-sites activity in which there is obligatory alternation of pyruvate carboxylating activity between pairs of subunits either face of the tetramer, does not occur in the hybrid tetramers. Our results are also discussed in relation to recent findings that there are multiple pathways of biotin carboxylation and decarboxylation between subunits in pyruvate carboxylase tetramers.

MicroRNA-143-3p targets pyruvate carboxylase expression and controls proliferation and migration of MDA-MB-231 cells.

Pyruvate carboxylase (PC) is a biotin-containing enzyme that converts pyruvate to oxaloacetate. We have previously shown that PC is overexpressed in highly invasive cancer cell lines where it supports biosynthesis during rapid cell growth. Here, we show that miR-143-3p suppresses the expression of PC in MDA-MB-231 cells by targeting its conserved binding site in the 3'-untranslated region (UTR) of human PC mRNA. Incorporation of the PC 3'UTR into a luciferase reporter gene inhibited expression of luciferase by 50% while mutation of the miR-143-3p binding site abrogated this inhibitory effect in MDA-MB-231 cells but not in low aggressive MCF-7 cell line. Transfection of miR-143-3p mimic or overexpression of miR-143-3p using tetracycline-inducible system in MDA-MB-231 cells down-regulated expression of both endogenous PC mRNA and protein by 40% and 50% respectively, confirming the regulatory role of miR-143-3p in PC expression. Induction of miR-143-3p expression at low and high levels lowered proliferation, metabolic activity and migration of MDA-MB-231 cells, in a dose-dependent manner. Re-expression of PC in MDA-MB-231 cells which were induced to express miR-143-3p partially restored migration but not proliferation, indicating that miR-143-3p regulates proliferation and migration through multiple pathways.

Differential contribution of pyruvate carboxylation to anaplerosis and cataplerosis during non-gluconeogenic and gluconeogenic conditions in HepG2 cells.

Pyruvate carboxylase (PC) is an anaplerotic enzyme that supplies oxaloacetate to mitochondria enabling the maintenance of other metabolic intermediates consumed by cataplerosis. Using liquid chromatography mass spectrometry (LC-MS) to measure metabolic intermediates derived from uniformly labeled 13C6-glucose or [3-13C]l-lactate, we investigated the contribution of PC to anaplerosis and cataplerosis in the liver cell line HepG2. Suppression of PC expression by short hairpin RNA lowered incorporation of 13C glucose incorporation into tricarboxylic acid cycle intermediates, aspartate, glutamate and sugar derivatives, indicating impaired cataplerosis. The perturbation of these biosynthetic pathways is accompanied by a marked decrease of cell viability and proliferation. In contrast, under gluconeogenic conditions where the HepG2 cells use lactate as a carbon source, pyruvate carboxylation contributed very little to the maintenance of these metabolites. Suppression of PC did not affect the percent incorporation of 13C-labeled carbon from lactate into citrate, α-ketoglutarate, malate, succinate as well as aspartate and glutamate, suggesting that under gluconeogenic condition, PC does not support cataplerosis from lactate.

miRNA Regulation of Glucose and Lipid Metabolism in Relation to Diabetes and Non-alcoholic Fatty Liver Disease.

Glucose and lipids are important nutrients because they provide most of the energy for the cells. A pre-translational regulation by microRNAs (miRNAs) plays a pivotal role in cellular metabolism by targeting the key rate-limiting enzymes of relevant pathways to fine-tune control of metabolic homeostasis. Aberrant expression of these miRNAs can result in an over or under expression of those key enzymes, contributing to the etiology of diabetes and non-alcoholic fatty liver disease (NAFLD). Here we discuss recent studies of various miRNAs that control insulin sensitivity, hepatic glucose production and de novo lipogenesis and how aberrant expression of these miRNAs contributes to the pathophysiology of diabetes and NAFLD in animal models. We also review the current application of circulating miRNAs as biomarkers for diagnosis or disease monitoring in diabetes and NAFLD.

Mass spectrometry analysis shows the biosynthetic pathways supported by pyruvate carboxylase in highly invasive breast cancer cells.

We recently showed that the anaplerotic enzyme pyruvate carboxylase (PC) is up-regulated in human breast cancer tissue and its expression is correlated with the late stages of breast cancer and tumor size [Phannasil et al., PloS One 10, e0129848, 2015]. In the current study we showed that PC enzyme activity is much higher in the highly invasive breast cancer cell line MDA-MB-231 than in less invasive breast cancer cell lines. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with 13C6-glucose and 13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction in viability and proliferation rates suggesting the perturbation of pathways that are involved in cancer invasiveness. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.

Investigation of the Roles of Allosteric Domain Arginine, Aspartate, and Glutamate Residues of Rhizobium etli Pyruvate Carboxylase in Relation to Its Activation by Acetyl CoA.

The mechanism of allosteric activation of pyruvate carboxylase by acetyl CoA is not fully understood. Here we have examined the roles of residues near the acetyl CoA binding site in the allosteric activation of Rhizobium etli pyruvate carboxylase using site-directed mutagenesis. Arg429 was found to be especially important for acetyl CoA binding as substitution with serine resulted in a 100-fold increase in the Ka of acetyl CoA activation and a large decrease in the cooperativity of this activation. Asp420 and Arg424, which do not make direct contact with bound acetyl CoA, were nonetheless found to affect acetyl CoA binding when mutated, probably through changed interactions with another acetyl CoA binding residue, Arg427. Thermodynamic activation parameters for the pyruvate carboxylation reaction were determined from modified Arrhenius plots and showed that acetyl CoA acts to decrease the activation free energy of the reaction by both increasing the activation entropy and decreasing the activation enthalpy. Most importantly, mutations of Asp420, Arg424, and Arg429 enhanced the activity of the enzyme in the absence of acetyl CoA. A main focus of this work was the detailed investigation of how this increase in activity occurred in the R424S mutant. This mutation decreased the activation enthalpy of the pyruvate carboxylation reaction by an amount consistent with removal of a single hydrogen bond. It is postulated that Arg424 forms a hydrogen bonding interaction with another residue that stabilizes the asymmetrical conformation of the R. etli pyruvate carboxylase tetramer, constraining its interconversion to the symmetrical conformer that is required for catalysis.

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

L-aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of L-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that L-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. L-aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of L-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of L-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. L-aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, L-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and L-aspartate) are spatially distinct but connected by a network of allosteric interactions.

Coordinating role of His216 in MgATP binding and cleavage in pyruvate carboxylase.

His216 is a well-conserved residue in pyruvate carboxylases and, on the basis of structures of the enzyme, appears to have a role in the binding of MgATP, forming an interaction with the 3'-hydroxyl group of the ribose ring. Mutation of this residue to asparagine results in a 9-fold increase in the Km for MgATP in its steady-state cleavage in the absence of pyruvate and a 3-fold increase in the Km for MgADP in its steady-state phosphorylation by carbamoyl phosphate. However, from single-turnover experiments of MgATP cleavage, the Kd of the enzyme·MgATP complex is essentially the same in the wild-type enzyme and H216N. Direct stopped-flow measurements of nucleotide binding and release using the fluorescent analogue FTP support these observations. However, the first-order rate constant for MgATP cleavage in the single-turnover experiments in H216N is only 0.75% of that for the wild-type enzyme, and thus, the MgATP cleavage step is rate-limiting in the steady state for H216N but not for the wild-type enzyme. Close examination of the structure of the enzyme suggested that His216 may also interact with Glu218, which in turn interacts with Glu305 to form a proton relay system involved in the deprotonation of bicarbonate. Single-turnover MgATP cleavage experiments with mutations of these two residues resulted in kinetic parameters similar to those observed in H216N. We suggest that the primary role of His216 is to coordinate the binding of MgATP and the deprotonation of bicarbonate in the reaction to form the putative carboxyphosphate intermediate by participation in a proton relay system involving Glu218 and Glu305.

Hepatocyte nuclear factor 4α regulates the expression of the murine pyruvate carboxylase gene through the HNF4-specific binding motif in its proximal promoter.

Pyruvate carboxylase (PC) is the first regulatory enzyme of gluconeogenesis. Here we report that the proximal promoter of the murine PC gene contains three binding sites for hepatocyte nuclear factor 4α (HNF4α). These sites include the classical direct repeat 1 (DR1) (-386/-374), non-perfect DR1 (-118/-106) and HNF4α-specific binding motif (H4-SBM) (-26/-14). Under basal conditions, mutation of the non-perfect DR1 decreased promoter activity by 50%, whereas mutation of neither the DR1 nor the H4-SBM had any effect. In marked contrast, only mutation of the H4-SBM decreased HNF4α-transactivation of the promoter activity by 65%. EMSA revealed that HNF4α binds to the DR1site and H4-SBM with similar affinity while it binds poorly to the non-perfect DR1. Interestingly, this non-perfect DR1 also coincides with two E-boxes. Mutation of the non-perfect DR1 together with the nearby E-box reduced USF1- but not USF2-transactivation of promoter activity, suggesting that USF1 partly contributes to the basal activity of the promoter. Substitution of the H4-SBM with the DR1 marginally reduced the basal promoter activity but did not eliminate HNF4α-transactivation, suggesting that HNF4α can exert its effect via DR1 within this promoter context. ChIP-assay confirmed that HNF4α is associated with the H4-SBM. Suppression of HNF4α expression in AML12 cells down-regulated PC mRNA and PC protein by 60% and 50%, respectively, confirming that PC is a target of HNF4α. We also propose a model for differential regulation of P1 promoter of PC gene in adipose tissue and liver.

Hep88 mAb-initiated paraptosis-like PCD pathway in hepatocellular carcinoma cell line through the binding of mortalin (HSPA9) and alpha-enolase.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most prevalent hepatic cancer worldwide. Currently, a targeted therapy via monoclonal antibodies (mAbs) specific to tumor-associated antigen is undergoing continual development in HCC treatment. METHODS: In this regard, after establishing and consequently exploring Hep88 mAb's tumoricidal effect on hepatocellular carcinoma cell line (HepG2 cell line), the Hep88 mAb's specific antigens from both membrane and cytoplasmic fractions of HepG2 cell line were identified by 2-D gel electrophoresis and western blot analysis. After in-gel digestion and subsequent analysis by liquid chromatography-mass spectrometry (LC-MS), mortalin (HSPA9) and alpha-enolase were identified. The recombinant proteins specific to Hep88 mAb were cloned and expressed in E. coli BL21(DE3). Moreover, alteration of HepG2 and Chang liver cell line after being induced by Hep88 mAb for 1-3 days was investigated using a transmission electron microscope. RESULTS: The result demonstrated that Hep88 mAb can bind to the recombinant mortalin (HSPA9) and alpha-enolase. In addition, the gradual appearing of mitochondria vacuolization and endoplasmic reticulum dilatation were observed. Those characteristics might be explained by the paraptosis-like program cell death (PCD), which is induced by the binding of Hep88 mAb to mortalin (HSPA9). Mortalin depletion resulting from the formation of Hep88 mAb-mortalin (HSPA9) complex might initiate transcription-independence of p53-mediated apoptosis. Additionally, Hep88mAb-alpha-enolase complex might initiate HepG2 cells energy exhaustion by glycolysis pathway obstruction. CONCLUSION: These fascinating results imply that Hep88 mAb might be a promising tool for the development of an effective treatment of HCC in the next decade.

Cellular signals integrate cell cycle and metabolic control in cancer.

Growth factors are the small peptides that can promote growth, differentiation, and survival of most living cells. However, aberrant activation of receptor tyrosine kinases by GFs can generate oncogenic signals, resulting in oncogenic transformation. Accumulating evidence support a link between GF/RTK signaling through the major signaling pathways, Ras/Erk and PI3K/Akt, and cell cycle progression. In response to GF signaling, the quiescent cells in the G0 stage can re-enter the cell cycle and become the proliferative stage. While in the proliferative stage, tumor cells undergo profound changes in their metabolism to support biomass production and bioenergetic requirements. Accumulating data show that the cell cycle regulators, specifically cyclin D, cyclin B, Cdk2, Cdk4, and Cdk6, and anaphase-promoting complex/cyclosome (APC/C-Cdh1) play critical roles in modulating various metabolic pathways. These cell cycle regulators can regulate metabolic enzyme activities through post-translational mechanisms or the transcriptional factors that control the expression of the metabolic genes. This fine-tune control allows only the relevant metabolic pathways to be active in a particular phase of the cell cycle, thereby providing suitable amounts of biosynthetic precursors available during the proliferative stage. The imbalance of metabolites in each cell cycle phase can induce cell cycle arrest followed by p53-induced apoptosis.

MicroRNA expression profiles associated with the metastatic ability of MDA­MB­231 breast cancer cells.

Breast cancer is an important worldwide public health concern. The incidence rate of breast cancer increases every year. The primary cause of death is metastasis, a process by which cancer cells spread from a primary site to secondary organs. MicroRNAs (miRs/miRNAs) are small non-coding RNAs that control gene expression at the post-transcriptional level. Dysregulation of certain miRNAs is involved in carcinogenesis, cancer cell proliferation and metastasis. Therefore, the present study assessed miRNAs associated with breast cancer metastasis using two breast cancer cell lines, the low-metastatic MCF-7 and the highly metastatic MDA-MB-231. miRNA array analysis of both cell lines indicated that 46 miRNAs were differentially expressed when compared between the two cell lines. A total of 16 miRNAs were upregulated in MDA-MB-231 compared with MCF-7 cells, which suggested that their expression levels may be associated with the highly invasive phenotype of MDA-MB-231 cells. Among these miRNAs, miR-222-3p was selected for further study and its expression was confirmed by reverse transcription-quantitative PCR (RT-qPCR). Under both non-adherent and adherent culture conditions, the expression levels of miR-222-3p in the MDA-MB-231 cell line were higher than those noted in the MCF-7 cell line under the same conditions. Suppression of endogenous miR-222-3p expression in MDA-MB-231 cells using a miR-222-3p inhibitor resulted in a 20-40% reduction in proliferation, and a ~30% reduction in migration, which suggested that the aggressive phenotype of MDA-MB-231 cells was partly regulated by miR-222-3p. Bioinformatic analysis of miR-222-3p using TargetScan 8.0, miRDB and PicTar identified 25 common mRNA targets, such as cyclin-dependent kinase inhibitor 1B, ADP-ribosylation factor 4, iroquois homeobox 5 and Bcl2 modifying factor. The results of the present study indicated that miR-222-3p was potentially associated with the proliferation and migratory ability of the MDA-MB-231 cell line.

Anti-Xanthine Oxidase 5'-Hydroxyhericenes A-D from the Edible Mushroom Hericium erinaceus and Structure Revision of 3-[2,3-Dihydroxy-4-(hydroxymethyl)tetrahydrofuran-1-yl]-pyridine-4,5-diol.

Hericium erinaceus is an edible mushroom with diverse pharmaceutical applications. Although this mushroom is an attractive source of natural products for cancer treatment, little is known about the bioactive compounds from this mushroom, which may possess antibreast cancer activity. Here, we report the isolation and structure elucidation of new compounds, 5'-hydroxyhericenes A-D (1-4) as an inseparable mixture, together with known compounds (5-16) from the fruiting body of H. erinaceus. Based on NMR spectroscopic data and MS fragmentation analysis, the structure of a previously reported natural product, 3-[2,3-dihydroxy-4-(hydroxymethyl)tetrahydrofuran-1-yl]-pyridine-4,5-diol (5), should be revised to adenosine (6). Compounds 1-4 inhibit xanthine oxidase activity, while compounds 6, 9, and 10 scavenge reactive oxygen species generated by xanthine oxidase. Moreover, hericerin (13) exhibits strong growth inhibitory activity against T47D breast cancer cells and, to a lesser extent, against MDA-MB-231 breast cancer and MRC-5 normal embryonic cells. Exposure of T47D and MDA-MB-231 cells slightly increased PARP cleavage, suggesting that the growth inhibitory effect of hericerin may be mediated through nonapoptotic pathways. Our results suggest that the bioactive compounds of mushroom H. erinaceus hold promise as antibreast cancer agents.

Proteomic analysis of holocarboxylase synthetase deficient-MDA-MB-231 breast cancer cells revealed the biochemical changes associated with cell death, impaired growth signaling, and metabolism.

We have previously shown that the holocarboxylase synthetase (HLCS) is overexpressed in breast cancer tissue of patients, and silencing of its expression in triple-negative cancer cell line inhibits growth and migration. Here we investigated the global biochemical changes associated with HLCS knockdown in MDA-MB-231 cells to discern the pathways that involve HLCS. Proteomic analysis of two independent HLCS knockdown cell lines identified 347 differentially expressed proteins (DEPs) whose expression change > 2-fold (p < 0.05) relative to the control cell line. GO enrichment analysis showed that these DEPs were mainly associated with the cellular process such as cellular metabolic process, cellular response to stimulus, and cellular component organization or biogenesis, metabolic process, biological regulation, response to stimuli, localization, and signaling. Among the 347 identified DEPs, 64 proteins were commonly found in both HLCS knockdown clones, confirming their authenticity. Validation of some of these DEPs by Western blot analysis showed that plasminogen activator inhibitor type 2 (SerpinB2) and interstitial collagenase (MMP1) were approximately 90% decreased in HLCS knockdown cells, consistent with a 50%-60% decrease in invasion ability of knockdown cells. Notably, argininosuccinate synthase 1 (ASS1), one of the enzymes in the urea cycle, showed approximately a 10-fold increase in the knockdown cells, suggesting the crucial role of HLCS in supporting the urea cycle in the triple-negative cancer cell line. Collectively, our proteomic data provide biochemical insights into how suppression of HLCS expression perturbs global changes in cellular processes and metabolic pathways, impairing cell growth and invasion.

Diminishing acetyl-CoA carboxylase 1 attenuates CCA migration via AMPK-NF-κB-snail axis.

Cholangiocarcinoma (CCA), a cancer of the biliary tract, is a significant health problem in Thailand. Reprogramming of cellular metabolism and upregulation of lipogenic enzymes have been revealed in CCA, but the mechanism is unclear. The current study highlighted the importance of acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme in de novo lipogenesis, on CCA migration. ACC1 expression in human CCA tissues was determined by immunohistochemistry. The results demonstrated that increased ACC1 was related to the shorter survival of CCA patients. Herein, ACC1-deficient cell lines (ACC1-KD) were generated by the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (cas9) system and were used for the comparative study. The ACC1 levels in ACC1-KD were 80-90 % lower than in parental cells. Suppression of ACC1 significantly reduced intracellular malonyl-CoA and neutral lipid contents. Two-fold growth retardation and 60-80 % reduced CCA cell migration and invasion were observed in ACC1-KD cells. The reduced 20-40 % of intracellular ATP levels, AMPK activation, lowered NF-κB p65 nuclear translocation, and snail expression were emphasized. Migration of ACC1-KD cells was restored by supplementation with palmitic acid and malonyl-CoA. Altogether, the importance of rate-limiting enzyme in de novo fatty acid synthesis, ACC1, and AMPK-NF-κB-snail axis on CCA progression was suggested herein. These might be the novel targets for CCA drug design. (ACC1, AMPK, Cholangiocarcinoma, De novo lipogenesis, NF-κB, Palmitic acid).