The edible mushroom Pleurotus citrinopileatus Singer contains bioactive compounds that may be used for the management of obesity. Despite extensive research, secondary metabolites with lipase inhibition and suppression of lipid accumulation remain unexplored. Herein, we report the isolation, structure elucidation, and biological evaluation of secondary metabolites from the fruiting bodies of P. citrinopileatus. Based on spectroscopic analysis and a comparison with spectroscopic data reported in the literatures, six compounds were identified: ergosterol (1), cerevisterol (2), uracil (3), a diastereomeric mixture of 5'-deoxy-5'-methylaminoadenosine (4), diethylene glycol monobutyl ether (5), and tryptophan (6). Compounds 1-6 showed mild lipase inhibitory activity ranging from 1.7 to 11.3%. In contrast to a previous report, ergosterol 1 did not inhibit lipid accumulation in 3T3-L1 adipocytes at 25, 50, and 75 µM. This finding provides valuable insight about secondary metabolites from the fruiting bodies of this mushroom and reports their lipase inhibitory and anti-lipid accumulation activities.

Mallotumide A, a cyclic heptapeptide from Mallotus spodocarpus previously noted for its anti-lipogenic properties in cancer models, was evaluated for its effect on 3T3-L1 adipogenesis. Cytotoxicity assays (24-48 h exposure) established an IC50 value of approximately 1 nM in preadipocytes. Notably, mature adipocytes maintained nearly 100% viability at concentrations from 0.001 to 1 nM, defining a nontoxic range to investigate the compound's modulation of lipid metabolism and differentiation. Treatment of preadipocytes with 1 nM Mallotumide A throughout the 10 day adipogenic differentiation process inhibited maturation into late-stage adipocytes and abolished lipid droplet accumulation. This effect was driven by the sustained downregulation of the master adipogenic regulators C/EBPα and PPARγ across both the early and late stages of differentiation. The disruption of these core transcriptional drivers subsequently reduced the expression of lipogenic enzymes, including pyruvate carboxylase, acetyl-CoA carboxylase 1, and fatty acid synthase, thereby preventing the maturation of the adipocytes. Mallotumide A also impaired growth signaling by reducing Akt protein. In contrast, stress-activated kinases, including JNK and p38, were not affected. The impaired differentiation was not attributed to apoptosis induction, as neither caspase-3 protein level nor caspase-3/7 activity was altered. The analysis of oxygen consumption profiles revealed that Mallotumide A did not significantly impair major mitochondrial bioenergetic parameters. However, in immature adipocytes, Mallotumide A moderately reduced mitochondrial spare respiratory capacity while maintaining normal basal and ATP-linked respiration. Collectively, these findings indicate that Mallotumide A predominantly targets both the early and late stages of differentiation. This effect is mediated through the downregulation of key adipogenic regulators and lipogenic enzymes, alongside the partial modulation of mitochondrial metabolism.

We have recently isolated, and determined the structure of a cycloheptapeptide, Mallotumide A from the Mallotus spodocarpus root extract. Here we reported the anti-cancer activity of Mallotumide A in highly invasive colon cancer, HCT116 and triple-negative breast cancer, MDA-MB-231 cell lines. Mallotumide A, at concentrations of 1 nM and 10 nM, completely inhibited the clonogenic growth, migration, and invasion of HCT116 and MDA-MB-231 cells, respectively. While the compound interfered with cell cycle progression without inducing apoptosis, exposure to 10 nM Mallotumide A for 48 h reduced the expression of two key lipogenic enzymes, ACC1 and FASN, by approximately 50% in both cell lines. The downregulation of ACC1 and FASN was accompanied by a 50% reduction in intracellular triglyceride levels while the cholesterol levels remained unaffected. Mallotumide A also moderately decreased AMP-activated protein kinase (AMPK) and ATP levels. Extracellular flux analysis revealed that acute exposure of both cancer cell lines to 1 nM and 10 nM Mallotumide A for 24 h markedly lowered the oxygen consumption rate. This was accompanied by reductions in basal and ATP-linked respiration, maximal respiration, and mitochondrial spare respiratory capacity. Mallotumide A also decreased the extracellular acidification rate, affecting both basal glycolysis and the glycolytic reserve. These findings suggest that the anti-cancer effects of Mallotumide A are associated with disruptions in cellular energy metabolism and the de novo lipogenesis pathway in cancer cells. This study underscores the potential of Mallotumide A as a novel anti-cancer agent.

Targeting cancer metabolism has become a promising strategy for cancer treatment. Pyruvate carboxylase (PC) is an anaplerotic enzyme that replenishes tricarboxylic acid cycle intermediates. PC is overexpressed in several types of human cancers, especially in aggressive cancers, including breast and colon cancer, where it promotes growth, survival, and metastasis. Recently, N4-((5-(4-(benzyloxy)phenyl)-2-thiophenyl)methyl)-N2-isobutyl-2,4-pyrimidinediamine (ZY-444) has been reported to possess anti-cancer activity by inhibiting PC activity in breast cancer. In this study, we expanded the potential of ZY-444 to highly aggressive colon cancer, HT-29 and HCT116, and further enhanced its efficacy using nanodelivery systems. The results reveal that ZY-444 not only inhibits the clonogenic growth of these colon cancer cells but also downregulates the expression of several key lipogenic enzymes, accompanied by a marked reduction in triglyceride and cholesterol levels. To improve its efficacy, ZY-444 was encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles with a surface coated with riboflavin (Rf)-functionalized chitosan (CSRf), a ligand known to target Rf transporters. The ZY-444-loaded PLGA nanoparticles exhibited a spherical morphology with an approximate size of 300 nm and a positive zeta potential, demonstrating their suitability for drug delivery applications. Compared with the free compound, ZY-444 encapsulated in nanoparticles demonstrated markedly enhanced efficacy being 11-, 14-, and 19-fold more potent in inhibiting the clonogenic growth of HT-29, HCT116, and MDA-MB-231 cells, respectively. In addition, the encapsulated form was over 10 times more effective at suppressing the migration and invasion of HCT116 and MDA-MB-231 cells. Encapsulated ZY-444 also more strongly inhibited the expression of lipogenic enzymes, further reinforcing its effect on lipid metabolism. Collectively, these findings underscore the potential of ZY-444-loaded PLGA nanoparticles as a promising drug delivery system for suppressing colon and breast cancer through targeted inhibition of the de novo lipogenesis pathway.

Erianin, a natural bibenzyl compound, has recently garnered attention owing to its diverse biological activities. In the present study, we investigated the effects of Erianin on adipocyte differentiation, lipid metabolism, and mitochondrial respiration in murine 3T3-L1 cells. Cytotoxicity assays indicated that Erianin exhibited low toxicity towards preadipocytes at concentrations up to 200 μM. Treatment with 20 μM Erianin completely inhibited the differentiation of 3T3-L1 preadipocytes into mature adipocytes and reduced lipid droplets. Western blot analysis revealed that Erianin attenuated Akt and p38 MAPK signalling without inducing apoptosis, suppressed the expression of key pro-adipogenic transcription factors, C/EBPα and PPARγ during the early stages of differentiation. This suppression was accompanied by the downregulation of lipogenic enzymes, including acetyl-CoA carboxylase 1 (ACC1), fatty acid synthase (FASN), pyruvate carboxylase (PC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR). While early-stage differentiation was robustly inhibited, higher concentrations (≥25 μM) were required to suppress terminal differentiation of immature adipocytes. This late-stage inhibition was accompanied by decreased expression of PPARγ, PC, and HMGCR, with minimal effects on ACC1 and FASN, suggesting a more modest role for Erianin in terminal adipogenesis. Assessment of mitochondrial metabolism of 3T3-L1 cells following 24-hour treatment of Erianin showed that it modestly impaired ATP-linked respiration, maximal respiration, spare respiratory capacity and intracellular ATP levels while basal respiration was unaffected. Collectively, these findings indicated that Erianin predominantly targets early adipogenic differentiation and mitochondrial bioenergetics.

In this work, we synthesize a quinoline-based heptamethine cyanine, QuCy7, with sulfonate groups to enhance water solubility. This dye demonstrates exceptional near-infrared absorption beyond 750 nm, accompanied by photothermal properties but low photostability. Encapsulating QyCy7 with polyethylene glycol to form nanopolymer, QuCy7@mPEG NPs, addresses the issue of its photoinstability. TEM showed that QuCy7@mPEG NPs possess a spherical morphology, featuring a core-shell structure with a size of around 120 nm in diameter. Upon irradiation with an 808 nm laser for 10 min, a significant increase in temperature up to 24 °C can be achieved with a photothermal conversion (PTC) rate of approximately 35%. QuCy7@mPEG NPs exhibit remarkable photothermal stability as compared to QuCy7. The efficiency of QuCy7@mPEG NPs was demonstrated by the in vitro PTT studies. Finally, the nanoparticles’ acute toxicity and effectiveness were assessed using the chick embryo model. The results provide compelling evidence that QuCy7@mPEG NPs are safe without inducing hemolysis, inhibit angiogenesis when exposed to light, and exhibit anti-tumor activity with a 76% reduction in tumor size compared to QuCy7 (40%). Thus suggesting the sulfonate groups can enhance water solubility, and its nanopolymer is biocompatible and possesses superior anti-tumor efficacy.

We have previously shown that the overexpression of acetyl-CoA carboxylase 1 (ACC1) was associated with the poor prognosis of cholangiocarcinoma (CCA) patients, and suppression of its expression in CCA cell lines deteriorated cell growth. The present study explored the mechanism by which ACC1 inhibition affects global protein acetylation, using genetic knockdown and pharmacological inhibition with an ACC1 inhibitor ND-646 as models. Both ACC1 knockdown and ACC1-inhibitor-treated cells displayed the hyperacetylation of proteins, accompanied by impaired growth and migration. The immunoprecipitation of hyperacetylated proteins using the anti-acetylated lysine antibody, followed by tandem mass spectrometry, identified three potential verification candidates, namely POTE ankyrin domain family member E, peroxisomal biogenesis factor 1, and heat shock protein 90 beta (HSP90B). HSP90 acetylation was the candidate selected for the verification of protein acetylation. To establish the effects of protein hyperacetylation, treatment with suberoylanilide hydroxamic acid (SAHA), a lysine deacetylase inhibitor, was conducted, and this served as an independent model. Decreased tumor growth but increased acetylated protein levels were observed in ACC1-KD xenograft tumors. Hyperacetylated-alleviated cell growth and migration were consistently observed in the SAHA-treated models. The molecular linkage between protein hyperacetylation and the AKT/GSK3β/Snail pathway was demonstrated. This study highlighted the importance of protein acetylation in CCA progression, suggesting that ACC1 and KDAC are potential targets for CCA treatment.

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.

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.

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.

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).

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.

Aquilaria crassna is a herbal plant that has recently been reported to possess several biological activities. A. crassna leaf extracts have been demonstrated to have a glucose-lowering effect in animal models. However, it is unclear what phytochemical compounds mediate this antidiabetic property. Here, we describe analytical methods for identifying such compounds from dried leaves by differential extractions with ethanol, butanol, ethyl acetate, and water, respectively. The phytochemical compounds in each fraction were further identified by gas chromatography-mass spectrometry. The cytotoxicity of these fractions was tested against a HepG2 cell line, while the rate of glucose utilization was determined using glucose oxidase assay. Lastly, the inhibitory effect on suppression of hepatic glucose production in HepG2 cells was determined by quantitative real-time PCR of genes encoding pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, glucose-6-phosphatase, and liver glycogen synthase.

Pyruvate carboxylase (PC) is an important anaplerotic enzyme that replenishes the tricarboxylic acid cycle (TCA) intermediates. It prevents the collapse of the TCA cycle upon its intermediates are removed during high anabolic demand. We have recently shown that overexpression of PC protein was associated with staging, metastasis and poor survival of colorectal cancer patients. Herein, we generated the PC knockout (PC KO) colon cancer cell lines, HT-29, by CRISPR-Cas9 technique, as a model to understand the role of this enzyme in colorectal cancer. The PC KO HT-29 cell lines had no detectable PC protein and did not show abnormal cellular or nuclear structures. However, PC KO HT-29 cells showed a 50-60% reduction in their growth rate and a 60-70% reduction in migration. The deficient growth phenotype of PC KO HT-29 cells was associated with apoptotic induction with no apparent cell cycle disruption following five days of growth. Down-regulation of key lipogenic enzymes, including acetyl-CoA carboxylase-1 and fatty acid synthase, was also associated with growth inhibition, suggesting that the de novo lipogenesis is impaired. Furthermore, PC KO HT-29 cells were 50% and 60% more sensitive to 5-fluorouracil and glutaminase inhibitor, CB-839, at their IC50 concentrations, respectively, following 48 h exposure. The increased cytotoxicity of CB-839 to PC KO HT-29 cells was associated with increased poly (ADP-ribose) polymerase cleavage. However, this was not observed with PC KO cells exposed to 5-fluorouracil, suggesting that PC KO HT-29 cells were prone to CB-839-induced apoptosis. Collectively, these findings indicate that ablation of PC expression in HT-29 cells disrupts the metabolic homeostasis of cells and inhibits proliferation and migration, accompanied by apoptotic induction. This study highlights the crucial role of PC in supporting the survival of HT-29 cells during exposure to chemotherapeutic drugs.

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.

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.

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.