Fatty acid metabolism in human breast cancer cells (MCF7) transfected with heart-type fatty acid binding protein

The human breast cancer cell line MCF7 does not express heart-type fatty acid binding protein (H-FABP), a marker protein for differentiated mammary gland. MCF7 cells transfected with the bovine H-FABP cDNA expressed the corresponding protein and were characterized by growth inhibition and lower tumorgenicity in nude mice [22]. By enzyme linked immunoassay we now determined the amount of bovine H-FABP in these cells as 638 ± 80 ng/mg protein and used the transfected cells to study the role of H-FABP in fatty acid metabolism. Compared to control cells the uptake of radioactively labelled palmitic acid and oleic acid into MCF7 cells after 30 or 60 min was increased by 67% in H-FABP expressing transfectants, demonstrating a stimulatory role for this FABP-type in fatty acid metabolism. However, preferential targeting of [¹⁴C]oleic acid into neutral or phospholipid classes was not observed by the criterion of high performance thin layer chromatography followed by autoradiography. A reason for the modest increase of fatty acid uptake in H-FABP transfected MCF7 cells may be the basal expression of epidermal-type FABP, which was detected for the first time in these cells. It appears that the small amount of E-FABP expressed in MCF7 cells fulfils the need of the cells for a cytosolic fatty acid carrier under culture conditions and that even high concentrations of another FABP do only slightly increase the uptake due to limitations of fatty acid transport through the plasma membrane or of metabolism.     

Fatty acid-binding protein and its relation to fatty acid oxidation

A relation between fatty acid oxidation capacity and cytosolic FABP content was found in heart and various muscles of the rat. Other tissues do not show such a relation, since they are involved in more or other pathways of fatty acid metabolism. At postnatal development FABP content and fatty acid oxidation capacity rise concomitantly in heart and quadriceps muscle in contrast to in liver and kidney. A dietary fat content of 40 en. % increased only the FABP content of liver and adipose tissue. Peroxisomal proliferators increased fatty acid oxidation in both liver and kidney, but only the FABP content of liver, and had no effect on heart and skeletal muscle. The FABP content of muscle did not show adaptation to various conditions. Only it increased in fast-twitch muscles upon chronic electrostimulation and endurance training.     

Chemical inhibition of fatty acid synthase: molecular docking analysis and biochemical validation in ocular cancer cells

Fatty acid biosynthesis is an attractive target for anti-cancer therapeutics. The ocular cancer, retinoblastoma cells were treated with fatty acid synthase (FASN) enzyme inhibitors: cerulenin, triclosan and orlistat. The IC₅₀ and dose-dependent sensitivity of cancer cells to FASN inhibitors decrease in biologic enzyme activity, and cell morphology alterations were analysed. Molecular interactions of enzyme-inhibitor complexes were studied by molecular modelling and docking simulations. The crystal structures of ketoacyl synthase (PDB ID:3HHD) (cerulenin) and thioesterase (PDB ID:2PX6) (orlistat) domains of human FASN were utilized for docking, while for the non-crystallised human FASN enoyl reductase domain (triclosan), homology model was built and used for docking. All three inhibitors showed significant binding energy indicating stable complex formation with their respective FASN subunits. The predicted Ki value of the FASN inhibitors corroborated well with their corresponding anti-cancer effects.     

Resveratrol suppresses the proliferation of breast cancer cells by inhibiting fatty acid synthase signaling pathway

In breast cancer cells, overexpression of human epidermal growth factor receptor 2 (HER2) increases the translation of fatty acid synthase (FASN) by altering the activity of PI3K/Akt signaling pathways. Cancer chemotherapy causes major side effects and is not effective enough in slowing down the progression of the disease. Earlier studies showed a role for resveratrol in the inhibition of FASN, but the molecular mechanisms of resveratrol-induced inhibition are not known. In the present study, we examined the novel mechanism of resveratrol on Her2-overexpressed breast cancer cells.The effect of resveratrol on the growth of breast cancer cells was assessed as percent cell viability by cytotoxicity-based MTT assay and the induction of apoptosis was determined by cell-death detection ELISA and flow cytometric analysis of Annexin-V–PI binding. Western immunobloting was used to detect signaling events in human breast cancer (SKBR-3) cells.Data showed that resveratrol-mediated down-regulation of FASN and HER2 genes synergistically induced apoptotic death in SKBR-3 cells. This concurrently caused a prominent up-regulation of PEA3, leads to down-regulation of HER2 genes. Resveratrol also alleviated the PI3K/Akt/mTOR signaling by down-regulation of Akt phosphorylation and up-regulation of PTEN expression.These findings suggest that resveratrol alters the cell cycle progression and induce cell death via FASN inhibition in HER2 positive breast cancer.     

Fatty acid oxidation and fatty acid synthesis in energy restricted rats

The importance of fat oxidation and fatty acid synthesis were examined in rats fed approximately one half their ad libitum food intake for a period of 13 days followed by 7 days of ad libitum feeding (refed rats). This study was undertaken because previous reports demonstrated that refed rats rapidly accumulated body fat. Our results confirmed this observation: refed rats accrued body fat and body weight at rates that were approximately 3 times higher than controls. Evidence for a period of increased metabolic efficiency was demonstrated by measuring the net energy requirement for maintenance over the refeeding period: refed rats had a reduced metabolic rate during the period of energy restriction (approximately 30% lower than control) and this persisted up to 2 days after the reintroduction of ad libitum feeding. The major factor responsible for the rapid fat gain was a depressed rate of fatty acid oxidation. Calculations of protein and carbohydrate intake over the refeeding period showed that the simplest explanation for the decrease in fatty acid oxidation is fat sparing. This is possible because of the large increase in dietary carbohydrate and protein intake during the refeeding period when metabolic rates are still depressed. The increased carbohydrate and protein may adequately compensate for the increasing energy requirements of the ER rats over the refeeding period affording rats the luxury of storing the excess dietary fat energy.     

Fatty acid synthase expression and esophageal cancer

Fatty acid synthase (FASN) overexpression has also been associated with a variety of human malignancies including tumor progression, aggressiveness, and metastasis. To investigate the role of FASN expression in esophageal cancer, we evaluated 60 cases of squamous cell carcinoma, 20 cases of adenocarcinoma, and 10 cases of normal esophageal tissues. We found that FASN was detected in 95?% human squamous cell carcinoma, and in 90?% human adenocarcinoma samples. However, all cases of normal esophageal epithelium did not express the protein of FASN. Further, to investigate the role of FASN in tumorigenesis and development, we analyze the growth and migration by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation and wound healing assay. We found that inhibition of FASN expression in TE13 cells by RNAi suppressed the growth of cells. Decreased FASN expression mitigated the migration of TE13 cells. These studies demonstrated the functional importance of FASN in esophageal tumorigenesis, and suggested that inhibiting FASN might be applied to treat esophageal cancer.     

Fatty acid oxidation by human liver peroxisomes.

A cyanide insensitive fatty acid oxidation system is detected in human liver and is shown to be localized in peroxisomes by subcellular fractionation in Metrizamide continuous density gradients. Fatty acyl-CoA oxidase, its characteristic enzyme, acts maximally on C₁₂–C₁₈ saturated fatty acids and on oleoyl-CoA and requires FAD. These results, together with the already established properties of the system in rat liver, support its potential contribution to lipid metabolism and to the hypolipidemic effect of Clofibrate and related drugs in humans.     

Induction of superoxide dismutase and cytotoxicity by manganese in human breast cancer cells.

MnCl2 induced manganese-containing superoxide dismutase (MnSOD) expression (mRNA, immunoreactive protein, and enzyme activity) in human breast cancer Hs578T cells. The induction of MnSOD immunoreactive protein in Hs578T cells was inhibited by tiron (a metal chelator and superoxide scavenger), pyruvate (a hydrogen peroxide scavenger), or 2-deoxy-d-glucose (DG, an inhibitor of glycolysis and the hexose monophosphate shunt), but not by 5,5-dimethyl-1-pyrroline-1-oxide (a superoxide scavenger), N-acetyl cysteine (a scavenger for reactive oxygen species and precursor of glutathione), diphenylene iodonium (an inhibitor of flavoproteins such as NADPH oxidase and nitric oxide synthase), or SOD (a superoxide scavenger). Northern blotting demonstrated that tiron or DG affected at the mRNA level, while pyruvate affected Mn-induced MnSOD expression at both the mRNA and protein levels. These results demonstrate that Mn can induce MnSOD expression in cultured human breast cancer cells. Mn also induced apoptosis and necrosis in these cells. Since inhibitors of Mn-induced MnSOD induction did not affect cell viability, MnSOD induction is probably not the cause of the Mn-induced cell killing.     

Effects of olive oil polyphenols on fatty acid synthase gene expression and activity in human colorectal cancer cells

Oleuropein (OL) and hydroxytyrosol (HT), the main olive oil polyphenols, possess anti-proliferative effects in vitro. Fatty acid synthase, a key anabolic enzyme of biosynthesis of fatty acids, plays an important role in colon carcinoma development. Our aim was to investigate whether gene expression of FAS, as well as its enzymatic activity, is regulated by HT and OL in two human colon cancer cell lines, as HT-29 and SW620. In addition, we investigated the effects of these polyphenols on growth and apoptosis in these cells. FAS gene expression and activity in treated HT-29 and SW620 cells were evaluated by real-time PCR and radiochemical assay, respectively. Cell growth and apoptosis, after polyphenols treatment, were measured by MTT test and flow cytometry, respectively. The inhibition of proliferation, detected after HT treatment, was mediated by an inhibition of FAS expression and its enzymatic activity in SW620 cells, while the anti-proliferative effect in HT-29 cells seems to be independent from FAS. OL exerted an anti-proliferative effect only on SW620 cells with a mechanism which excluded FAS. Olive oil polyphenols used were able to induce apoptosis in both cell lines studied. The increase of apoptosis in these cells was accompanied by the block of cell cycle in the S phase. This study demonstrates that HT and OL may induce anti-proliferative and pro-apoptotic effects only in certain human colorectal cancer cell types. These effects are FAS mediated only in SW620 cells after treatment with HT.     

Effects of anti-proliferative lichen metabolite,protolichesterinic acid on fatty acid synthase,cell signalling and drug response in breast cancer cells

BackgroundThe lichen compound (+)-protolichesterinic acid (+)-PA, isolated from Iceland moss, has anti-proliferative effects on several cancer cell lines. The chemical structure of (+)-PA is similar to a known fatty acid synthase (FASN) inhibitor C75.AimsTo test whether the anti-proliferative activity of (+)-PA is associated with effects on FASN and HER2 (human epidermal growth factor receptor 2) and major signalling pathways. Synergism between (+)-PA and lapatinib, a HER2 active drug, was also evaluated.Materials and methodsPure compound was isolated by preparative high-performance liquid chromatography (HPLC) and purity of (+)-PA analyzed by analytical HPLC. Cell viability was assessed using Crystal violet staining. FASN and HER2 expression was estimated by immunofluorescence. The Meso Scale Discovery (MSD)^® assay was used to measure activation of ERK1/2 and AKT. Synergism was estimated by the CalcuSyn software.ResultsTreatment with (+)-PA increased FASN expression in SK-BR-3 cells, which overexpress FASN and HER2, implying a compensatory response to inhibition of FASN activity. HER2 expression was decreased suggesting secondary downregulation. ERK1/2 and AKT signalling pathways were inhibited, probably due to reduced levels of HER2. No effects were observed in T-47D cells. Synergism between (+)-PA and lapatinib was observed in the SK-BR-3 cells.ConclusionResults suggest that the primary effect of (+)-PA is inhibition of FASN activity. Synergistic effects with lapatinib were seen only in SK-BR-3 cells, and not T-47D cells, further supporting the notion that (+)-PA acts by inhibiting FASN with secondary effects on HER2 expression and signalling. (+)-PA could therefore be a suitable agent for further testing, alone or in combination treatment against HER2-overexpressing breast cancer.     

Up-regulation of acetyl-CoA carboxylase alpha and fatty acid synthase by human epidermal growth factor receptor 2 at the translational level in breast cancer cells

Expression of the HER2 oncogene is increased in approximately 30% of human breast carcinomas and is closely correlated with the expression of fatty acid synthase (FASN). In the present study, we determined the mechanism by which FASN and acetyl-CoA carboxylase alpha (ACCalpha) could be induced by HER2 overexpression. SK-BR-3 and BT-474 cells, breast cancer cells that overexpress HER2, expressed higher levels of FASN and ACCalpha compared with MCF-7 and MDA-MB-231 breast cancer cells in which HER2 expression is low. The induction of FASN and ACCalpha in BT474 cells were not mediated by the activation of SREBP-1. Exogenous HER2 expression in MDA-MB-231 cells induced the expression of FASN and ACCalpha, and the HER2-mediated increase in ACCalpha and FASN was inhibited by both LY294002, a phosphatidylinositol 3-kinase inhibitor, and rapamycin, a mammalian target of rapamycin (mTOR) inhibitor. In addition, the activation of mTOR by the overexpression of RHEB in MDA-MB-231 cells increased the synthetic rates of both FASN and ACCalpha. On the other hand, FASN and ACCalpha were reduced in BT-474 cells by a blockade of the mTOR signaling pathway. These changes observed in their protein levels were not accompanied by changes in their mRNA levels. The 5'- and 3'-untranslated regions of both FASN and ACCalpha mRNAs were involved in selective translational induction that was mediated by mTOR signal transduction. These results strongly suggest that the major mechanism of HER2-mediated induction of FASN and ACCalpha in the breast cancer cells used in this study is translational regulation primarily through the mTOR signaling pathway.     

Nuclear factor-kappaB activation leads to down-regulation of fatty acid oxidation during cardiac hypertrophy

Little is known about the mechanisms responsible for the fall in fatty acid oxidation during the development of cardiac hypertrophy. We focused on the effects of nuclear factor (NF)-kappaB activation during cardiac hypertrophy on the activity of peroxisome proliferator-activated receptor (PPAR) beta/delta, which is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. Phenylephrine-induced cardiac hypertrophy in neonatal rat cardiomyocytes caused a reduction in the expression of pyruvate dehydrogenase kinase 4 (Pdk4), a target gene of PPARbeta/delta involved in fatty acid utilization, and a fall in palmitate oxidation that was reversed by NF-kappaB inhibitors. Lipopolysaccharide stimulation of NF-kappaB in embryonic rat heart-derived H9c2 myotubes, which only express PPARbeta/delta, caused both a reduction in Pdk4 expression and DNA binding activity of PPARbeta/delta to its response element, effects that were reversed by NF-kappaB inhibitors. Coimmunoprecipitation studies demonstrated that lipopolysaccharide strongly stimulated the physical interaction between the p65 subunit of NF-kappaB and PPARbeta/delta, providing an explanation for the reduced activity of PPARbeta/delta. Finally, we assessed whether this mechanism was present in vivo in pressure overload-induced cardiac hypertrophy. In hypertrophied hearts of banded rats the reduction in the expression of Pdk4 was accompanied by activation of NF-kappaB and enhanced interaction between p65 and PPARbeta/delta. These results indicate that NF-kappaB activation during cardiac hypertrophy down-regulates PPARbeta/delta activity, leading to a fall in fatty acid oxidation, through a mechanism that involves enhanced protein-protein interaction between the p65 subunit of NF-kappaB and PPARbeta/delta.     

Fatty acid metabolism in Paramecium. Oleic acid metabolism and inhibition of polyunsaturated fatty acid synthesis by triparanol

Paramecium requires oleic acid for growth and can grow in media containing no other fatty acids. In the present study, we have shown that this ciliate utilized oleate mainly as a carbon and energy source, even though this fatty acid was the only substrate available for synthesis of polyunsaturated fatty acids. Culture growth was inhibited by the addition of the drug triparanol. Triparanol decreased the formation of polyunsaturated fatty acids from oleate by preventing desaturation to form the dienoic acid, linoleate. Triparanol inhibition resulted in an altered phospholipid fatty acyl composition, an increased fragility and an altered behavioral response of the cells to a depolarizing stimulation solution. Therefore, although most of the dietary oleate was not used by the cells for polyunsaturated fatty acid synthesis, the desaturation of oleic acid was critical for normal culture growth, cell integrity and swimming behavior, all of which are expected to be dependent on normal membrane lipid composition.     

Deficiency in fatty acid synthase leads to premature cell death and dramatic alterations in plant morphology

An Arabidopsis mosaic death1 (mod1) mutant, which has premature cell death in multiple organs, was isolated. mod1 plants display multiple morphological phenotypes, including chlorotic and curly leaves, distorted siliques, premature senescence of primary inflorescences, reduced fertility, and semidwarfism. The phenotype of the mod1 mutant results from a single nuclear recessive mutation, and the MOD1 gene was isolated by using a map-based cloning approach. The MOD1 gene encodes an enoyl-acyl carrier protein (ACP) reductase, which is a subunit of the fatty acid synthase complex that catalyzes de novo synthesis of fatty acids. An amino acid substitution in the enoyl-ACP reductase of the mod1 mutant causes a marked decrease in its enzymatic activity, impairing fatty acid biosynthesis and decreasing the amount of total lipids in mod1 plants. These results demonstrate that a deficiency in fatty acid biosynthesis has pleiotropic effects on plant growth and development and causes premature cell death.     

Lipid metabolism and liver inflammation. II. Fatty liver disease and fatty acid oxidation

Fatty liver disease (FLD), whether it is alcoholic FLD (AFLD) or nonalcoholic FLD (NAFLD), encompasses a morphological spectrum consisting of hepatic steatosis (fatty liver) and steatohepatitis. FLD has the inherent propensity to progress toward the development of cirrhosis and hepatocellular carcinoma. It is generally difficult to distinguish AFLD from NAFLD on morphological grounds alone despite the distinctions implied by these etiological designations. The indistinguishable spectrum of histological features of both AFLD and NAFLD suggests a possible convergence of pathogenetic mechanisms at some critical juncture that enables the progression of steatohepatitis toward cirrhosis and liver cancer. From a pathogenetic perspective, FLD may be considered a single disease with multiple etiologies. Excess energy consumption and reduced energy combustion appear to be critical events that culminate in lipid storage in the liver. Energy combustion in the liver is controlled by peroxisome proliferator-activated receptor (PPAR)-alpha-regulated mitochondrial and peroxisomal fatty acid beta-oxidation systems and the microsomal omega-oxidation system. PPAR-alpha, a receptor for peroxisome proliferators, functions as a sensor for fatty acids (lipid sensor), and ineffective PPAR-alpha sensing can lead to reduced energy burning resulting in hepatic steatosis and steatohepatitis. Delineation of the pathogenetic aspects of FLD is necessary for developing novel therapeutic strategies for this disease.     

Altering bioelectricity on inhibition of human breast cancer cells

Background

Membrane depolarization is associated with breast cancer. Depolarization-activated voltage-gated ion channels are directly implicated in the initiation, proliferation, and metastasis of breast cancer.

Methods

In this study, the role of voltage-gated potassium and calcium ion channel modulation was explored in two different invasive ductal human carcinoma cell lines, MDA-MB-231 (triple-negative) and MCF7 (estrogen-receptor-positive).

Results

Resting membrane potential is more depolarized in MCF7 and MDA-MB-231 cells compared to normal human mammary epithelial cells. Increasing extracellular potassium concentration up to 50 mM depolarized membrane potential and greatly increased cell growth. Tetraethylammonium (TEA), a non-specific blocker of voltage-gated potassium channels, stimulated growth of MCF7 cells (control group grew by 201 %, 1 mM TEA group grew 376 %). Depolarization-induced calcium influx was hypothesized as a requirement for growth of human breast cancer. Removing calcium from culture medium stopped growth of MDA and MCF7 cells, leading to cell death after 1 week. Verapamil, a blocker of voltage-gated calcium channels clinically used in treating hypertension and coronary disease, inhibited growth of MDA cells at low concentration (10–20 μM) by 73 and 92 % after 1 and 2 days, respectively. At high concentration (100 μM), verapamil killed >90 % of MDA and MCF7 cells after 1 day. Immunoblotting experiments demonstrated that an increased expression of caspase-3, critical in apoptosis signaling, positively correlated with verapamil concentration in MDA cells. In MCF7, caspase-9 expression is increased in response to verapamil.

Conclusions

Our results support our hypotheses that membrane depolarization and depolarization-induced calcium influx stimulate proliferation of human breast cancer cells, independently of cancer subtypes. The underlying mechanism of verapamil-induced cell death involves different caspases in MCF7 and MDA-MB-231. These data suggest that voltage-gated potassium and calcium channels may be putative targets for pharmaceutical remediation in human invasive ductal carcinomas.

     

Cardiomyocyte-restricted peroxisome proliferator-activated receptor-delta deletion perturbs myocardial fatty acid oxidation and leads to cardiomyopathy

Fatty acid oxidation (FAO) is a primary energy source for meeting the heart's energy requirements. Peroxisome proliferator-activated receptor-delta (PPAR-delta) may have important roles in FAO. But it remains unclear whether PPAR-delta is required for maintaining basal myocardial FAO. We show that cre-loxP-mediated cardiomyocyte-restricted deletion of PPAR-delta in mice downregulates constitutive expression of key FAO genes and decreases basal myocardial FAO. These mice have cardiac dysfunction, progressive myocardial lipid accumulation, cardiac hypertrophy and congestive heart failure with reduced survival. Thus, chronic myocardial PPAR-delta deficiency leads to lipotoxic cardiomyopathy. Together, our data show that PPAR-delta is a crucial determinant of constitutive myocardial FAO and is necessary to maintain energy balance and normal cardiac function. We suggest that PPAR-delta is a potential therapeutic target in treating lipotoxic cardiomyopathy and other heart diseases.     

Fatty acid synthase (FASN) regulates the mitochondrial priming of cancer cells

Inhibitors of the lipogenic enzyme fatty acid synthase (FASN) have attracted much attention in the last decade as potential targeted cancer therapies. However, little is known about the molecular determinants of cancer cell sensitivity to FASN inhibitors (FASNis), which is a major roadblock to their therapeutic application. Here, we find that pharmacological starvation of endogenously produced FAs is a previously unrecognized metabolic stress that heightens mitochondrial apoptotic priming and favors cell death induction by BH3 mimetic inhibitors. Evaluation of the death decision circuits controlled by the BCL-2 family of proteins revealed that FASN inhibition is accompanied by the upregulation of the pro-death BH3-only proteins BIM, PUMA, and NOXA. Cell death triggered by FASN inhibition, which causally involves a palmitate/NADPH-related redox imbalance, is markedly diminished by concurrent loss of BIM or PUMA, suggesting that FASN activity controls cancer cell survival by fine-tuning the BH3 only proteins-dependent mitochondrial threshold for apoptosis. FASN inhibition results in a heightened mitochondrial apoptosis priming, shifting cells toward a primed-for-death state “addicted” to the anti-apoptotic protein BCL-2. Accordingly, co-administration of a FASNi synergistically augments the apoptosis-inducing activity of the dual BCL-X_L/BCL-2 inhibitor ABT-263 (navitoclax) and the BCL-2 specific BH3-mimetic ABT-199 (venetoclax). FASN inhibition, however, fails to sensitize breast cancer cells to MCL-1- and BCL-X_L-selective inhibitors such as {"type":"entrez-nucleotide","attrs":{"text":"S63845","term_id":"400540"}}S63845 and A1331852. A human breast cancer xenograft model evidenced that oral administration of the only clinically available FASNi drastically sensitizes FASN-addicted breast tumors to ineffective single-agents navitoclax and venetoclax in vivo. In summary, a novel FASN-driven facet of the mitochondrial priming mechanistically links the redox-buffering mechanism of FASN activity to the intrinsic apoptotic threshold in breast cancer cells. Combining next-generation FASNis with BCL-2-specific BH3 mimetics that directly activate the apoptotic machinery might generate more potent and longer-lasting antitumor responses in a clinical setting.Subject terms: Cancer metabolism, Lipid signalling     

The effects of inhibition of fatty acid oxidation in suckling newborn rats.

Inhibition of fatty acid oxidation with pent-4-enoate in suckling newborn rats caused a fall in blood [glucose] and blood [ketone bodies] and inhibition of gluconeogenesis from lactate. Glucose utilization was not increased in newborn rats injected with pent-4-enoate. Active fatty acid oxidation appears to be essential to support gluconeogenesis and to maintain normal blood [glucose] in suckling newborn rats.