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. 2025 Aug 27;10(35):40392–40401. doi: 10.1021/acsomega.5c05729

Inhibitory Effects and the Potential Mechanism of Phloretin on Animal Fatty Acid Synthase

Tongchun Lan 1, Xinru Zhang 1, Jiarui Xie 1, Xu Xing 1, Chunyan Wang 1, Xiaofeng Ma 1,*
PMCID: PMC12423902  PMID: 40949263

Abstract

Fatty acid synthase (FASN), a critical enzyme involved in lipid biosynthesis, is highly expressed in adipocytes and exhibits aberrant activity in diverse human cancers. Phloretin, a natural dihydrochalcone abundant in apple peels, crabapples, strawberries, and pears, has emerged as a potential modulator of FASN activity. This study investigates phloretin’s inhibitory effects on FASN and the underlying mechanisms. Biochemical assays revealed that phloretin inhibited FASN in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of 4.90 ± 0.66 μM. Kinetic analysis demonstrated distinct inhibition patterns: competitive inhibition against acetyl-CoA, mixed competitive/noncompetitive inhibition toward malonyl-CoA, and uncompetitive inhibition relative to NADPH. Molecular docking simulations further indicated that phloretin binds to the β-ketoacyl synthase (KS) domain of FASN, suggesting a mechanism distinct from that of typical flavonoid inhibitors. Notably, phloretin exhibited irreversible inhibition of FASN, in contrast with the inhibition observed for other flavonoids. To validate the cellular relevance, we demonstrated that phloretin suppressed FASN expression and enzymatic activity in breast cancer cells, concomitant with significant reductions in intracellular triglyceride (TG) accumulation and cancer cell viability. Although adipocytes were not studied in this work due to the long differentiation period required, future studies are planned to investigate FASN inhibition in adipogenesis models. Given FASN’s dual role as a therapeutic target in obesity and oncogenesis, these findings highlight phloretin’s translational potential as a multitarget agent for metabolic and neoplastic disorders. The well-characterized inhibition mechanism of phloretin, combined with its dual capacity to suppress lipogenesis and inhibit proliferation, establishes this natural compound as a compelling candidate for advanced preclinical evaluation in therapeutics for metabolic disorders.

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1. Introduction

Fatty acid synthase (FASN, EC 2.3.1.85) catalyzes the synthesis of saturated long-chain free fatty acids, predominantly palmitate, from acetyl-CoA (Ac-CoA) and malonyl-CoA (Mal-CoA) as precursors, with NADPH present as a cofactor. , FASN is a multifunctional enzyme complex that consists of two identical subunits, each of which contains seven independent functional domains: dehydrase (DH), enoyl reductase (ER), β-ketoacyl reductase (KR), β-ketoacyl synthase (KS), malonyl/acetyltransferase (MAT), thioesterase (TE), and the acyl carrier protein (ACP). Most human tissues exhibit low levels of FASN expression. However, the expression levels of FASN in human adipocytes and cancer cells are high. , In adipocytes and cancer cells, FASN plays an important role in cell proliferation, lipid deposition, progression, invasion, and metastasis. − According to the previous studies, FASN inhibitors such as cerulenin, C75, and epigallocatechin gallate (EGCG) induce weight loss, inhibit preadipocyte proliferation, and induce apoptosis in cancer cells. − Considering this, FASN has been considered a potential dual target for antiobesity and anticancer therapies. −

There is still a lack of clinically effective drugs to prevent or treat obesity or cancer. Food extracts or natural products isolated from food have been investigated extensively for preventing or ameliorating obesity and cancer. Phloretin, also known as β-(4-hydroxyphenyl)-2,4,6-trihydroxypropiophenone, is a dihydrochalcone that is found mainly in apple, crabapple, strawberry, and pear trees. Phloretin stimulates adipocyte lipolysis, prevents obesity in mice, and has been reported to exert anticancer effects in various cell types. Experimental studies have demonstrated antioxidant, anti-inflammatory, antituberculosis, anticancer, and antiobesity properties of phloretin. − Previous studies have found that phloretin can prevent weight gain, regulate blood glucose, improve mitochondrial dysfunction, and attenuate hepatic steatosis in high-fat diet-induced obese mice. , It was reported that phloretin remarkably reduced excessive lipid accumulation and decreased sterol regulatory element-binding protein 1c, blocking the expression of FASN in oleic acid-induced HepG2 cells. Phloretin has proven to improve insulin sensitivity and enhance glucose uptake in differentiated adipocytes. Phloretin could ameliorate hepatic steatosis through the regulation of lipogenesis. However, the effect of phloretin on adipogenesis has also been reported. Phloretin, as a glucose transporter inhibitor, promoted lipid accumulation by significantly increasing the expression of several adipogenic markers, including PPARγ, C/EBPα, FASN, fatty acid-binding protein 4, and adiponectin. Moreover, phloretin has emerged as a promising anticancer agent. Phloretin has been reported to reduce cell viability, inhibit proliferation and migration, induce apoptosis, suppress progression, inhibit autophagy, and arrest the cell cycle in a variety of human cancer cells. − To date, the precise mechanisms underlying phloretin’s anticancer and antiobesity activities remain unclear. In the present study, we evaluated the inhibitory effect of phloretin on animal FASN. Furthermore, the possible inhibitory mechanism of phloretin was investigated.

2. Materials and Methods

2.1. Chemicals

Phloretin (98% HPLC), Ac-CoA (93% HPLC), acetoacetyl-CoA (AcAc-CoA, 93% HPLC), Mal-CoA (90% HPLC), ethyl acetoacetate (98% GC), NADPH (98% HPLC), ethyl crotonate (98% GC), dithiothreitol (DTT, 98% HPLC), ethylenediaminetetraacetic acid (EDTA, 99%), and dimethyl sulfoxide (DMSO, 99% HPLC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were of analytical grade and were obtained from local suppliers.

2.2. Isolation and Purification of FASN

The FASN was prepared as previously described by isolating it from chicken liver using ammonium sulfate precipitation and column chromatography following standard protocols. The amino acid sequence of chicken FASN has 63% identity with that of the human enzyme. Homogenate supernatant (HSS) was prepared from freshly excised chicken livers by homogenization in ice-cold extraction buffer (100 mM K-Pi pH 7.8, 1 mM EDTA, and 1 mM DTT) at a 1:1.8 (w/v) tissue-to-buffer ratio. The homogenate was sequentially centrifuged (8,000 × g, 30 min; 35,000 × g, 30 min at 4 °C), and the clarified HSS was stored at −70 °C. For purification, HSS underwent two-step ammonium sulfate precipitation: 0.33 volumes of saturated ammonium sulfate (with freshly added 1 mM DTT) were added to the supernatant, followed by centrifugation (10,000 × g, 15 min, 4 °C). The resulting supernatant was treated with 0.25 volumes of saturated ammonium sulfate/DTT and recentrifuged. The pellet was discarded after FASN activity was confirmed to remain in the supernatant.

The active fraction was redissolved in 5 mM K-Pi buffer (pH 7.0, 1 mM DTT), adjusted to a conductivity equivalent to ≤50 mM K-Pi, and loaded onto a DEAE-cellulose column pre-equilibrated with the same buffer. Bound proteins were eluted by using a step gradient: FAS was recovered in the 0.16 M K-Pi/DTT fraction after washing with 50 mM K-Pi/DTT. The FASN-enriched pool was applied to a Blue-Sepharose column equilibrated with 50 mM K-Pi/DTT. FASN was eluted with 0.35 M NaCl in 50 mM K-Pi/DTT, while impurities were removed with 2 M NaCl/DTT.

The FASN peak was concentrated by adding 0.66 volumes of saturated ammonium sulfate/DTT, followed by centrifugation (10,000 × g, 15 min, 4 °C). The pellet was dissolved in storage buffer (100 mM K-Pi pH 7.0, 10 mM EDTA, 2% glycerol, 1 mM DTT) to a final concentration of 10 mg/mL and stored at −70 °C. All steps prior to DEAE chromatography were performed at 0–4 °C, with DTT added fresh to buffers. FASN activity was monitored spectrophotometrically at each stage.

2.3. Assays of FASN Activity and FASN Inhibition

The overall FASN reaction, β-ketoacyl reduction, enoyl reduction, and reduction of AcAc-CoA activities were all assessed at 37 °C by monitoring the decrease in NADPH absorbance at 340 nm. Four domains, including MAT, KR, DH, and ER, as well as the ACP of FASN, were involved in AcAc-CoA reduction. After equilibration at 37 °C for 10 min, the reaction was initiated by adding FASN to the reaction mixture. The initial velocity was calculated based on the decrease in absorbance at 340 nm.

After the addition of phloretin, the last substrate of FASN was added to the reaction system to initiate the reaction and determine the reversible inhibition. The FASN activities with or without phloretin were designated as A i and A 0, respectively. The value of A i/A 0 × 100% was defined as the relative activity. The half-inhibitory concentration (IC50) value was yielded from a plot of relative activity versus phloretin concentration. In order to avoid solvent interference, the largest amount of DMSO added to the reaction system was less than 0.5% (v/v) in each experiment.

The time-dependent inhibition, typically indicative of irreversible binding, was assessed as previously described. The FASN solution was mixed with various concentrations of phloretin and incubated at 25 °C. Subsequently, we measured the residual activity of the aliquot samples at specified intervals to obtain the time course. The inactivation rate constant can be calculated from the semi-logarithmic diagram of the time process, which is based on the calculation formula Ln A t /A 0 = −k obs t. A t /A 0 expresses the residual activity at time t, and k obs represents the observed rate constant.

2.4. Enzyme Kinetics Study

The possible inhibitory effects of phloretin on different domains of FASN were examined by keeping the phloretin concentration at several fixed levels and changing one substrate’s concentration while keeping the concentrations of the other two substrates unchanged. Lineweaver–Burk (double reciprocal) plots were generated at each phloretin concentration to evaluate the nature of inhibition with respect to each substrate.

2.5. Autodock and Molecular Dynamics

Semiflexible molecular docking was performed using the Lamarckian Genetic Algorithm (LGA) in AutoDock (version 4.2.6). The crystal structure of the human FASN KS domain (PDB ID: 3HHD) and the ligands phloretin (CID: 4788) and C75 (CID: 4248455) were retrieved from the Protein Data Bank and PubChem, respectively. PyMOL (version 2.6.0) was applied to remove water and other small molecules from the receptor and convert the format to PDB. AutoDockTools (version 1.5.6) was used to add the atom type to the receptor and polar hydrogen atoms and Gasteiger charges. The structures of both ligands and receptor proteins were then converted to the PDBQT format. Then, we selected a rigid receptor and flexible ligand and set the output number of genetic algorithm conformation searches to 100. Finally, we used PyMOL and Maestro (version 12.9) for visualization.

To further explore protein–ligand interactions, the most favorable docking pose was selected as the starting structure for molecular dynamics (MD) simulations using GROMACS 2020.3. Protein and ligand files were separated; ligand parameters were generated using the GAFF force field, and receptor parameters were assigned using the AMBER99SB force field. The system was solvated in a cubic TIP3P water box and neutralized with sodium ions. After energy minimization (10,000 steps, steepest descent algorithm), the system underwent 500 ps of NVT and NPT equilibration, followed by 100 ns of unrestrained MD simulation.

2.6. Cell Line and Cultures

Human breast cancer cell lines MDA-MB-231 and MCF-7 were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. Cells were grown in DMEM, supplemented with 10% FBS, and maintained in a humidified incubator containing 95% air and 5% CO2 at 37 °C.

2.7. Cell Viability Assay

Cell viability was measured using a Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan). Cells were seeded in 96-well plates at a density of 1 × 106 cells/mL and allowed to attach overnight. After treatment, the medium was replaced with a drug-free medium containing 10 μL of CCK-8 solution per well. Plates were incubated for 1 h at 37 °C, and absorbance was measured at 450 nm using a microplate reader (Multiskan MK3). All experiments were carried out six times on three different occasions.

2.8. Western Blot Analysis

Following the treatment of breast cancer cells with a corresponding concentration of phloretin for 24 h, the culture medium in the six-well plates was removed. The cells were gently washed with phosphate-buffered saline (PBS) and lysed using RIPA lysis buffer (P0013B, Beyotime). The lysates were collected in EP tubes, sonicated to disrupt cell membranes, and centrifuged to obtain the protein supernatants. Protein concentrations were quantified using a BCA Protein Assay Kit (20201ES86, Yeasen). Equal amounts of protein (20 μg per lane) were separated by 8% SDS-PAGE according to molecular weight and transferred to 0.45-μm pore size PVDF membranes (IPVH00010, Merck Millipore). The membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies: rabbit monoclonal anti-FASN (3180S) and anti-β-actin (4970S) (both from Cell Signaling Technology). Subsequently, the membranes were incubated with a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (H + L) secondary antibody (S0101, Lablead Biotech). Protein bands were visualized using an ECL detection kit (36208ES60, Yeasen). To quantify protein expression, band intensities were normalized to β-actin and analyzed by using ImageJ software. All experiments were carried out at least three times.

2.9. Intracellular FASN Activity Assay

After 24 h of exposure to phloretin, cells were harvested by trypsinization, pelleted by centrifugation, washed three times, and resuspended in cold PBS. Cells were sonicated at 4 °C and centrifuged at 12,000 g for 15 min at 4 °C to obtain particle-free supernatants. The FASN activity was determined spectrophotometrically by measuring the decrease of absorbance at 340 nm due to the oxidation of NADPH. A 50 μL particle-free supernatant, 25 mM KH2PO4–K2HPO4 buffer, 0.25 mM EDTA, 0.25 mM dithiothreitol, 30 μM Ac-CoA, and 350 μM NADPH (pH 7.0) in a total volume of 500 μL were monitored at 340 nm for 60 s to measure background NADPH oxidation. After the addition of 100 mM Mal-CoA, the reaction mixture was assayed for an additional 60 s to determine the FAS-dependent oxidation of NADPH.

2.10. Triglyceride (TG) Content Measurement

Cells were seeded in 10 cm culture dishes for 24 h, followed by phloretin treatment for an additional 24 h prior to TG quantification. After trypsin-EDTA digestion, the cells were collected and centrifuged to pellet the cell mass. The pellet was washed twice with ice-cold PBS and resuspended in PBS. The cell suspension was sonicated on ice to disrupt the membranes. Subsequently, reaction buffer (prepared according to the TG assay kit instructions) was added, and the mixture was incubated at 37 °C for 10 min. Absorbance at 500 nm was measured to calculate the TG content. Parallel samples were subjected to a BCA protein assay for normalization against total cellular protein concentration.

2.11. Statistical Analysis

Data were obtained from the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) and Tukey’s posttest were performed by Origin 8.5 software (OriginLab, MS, USA) to determine the statistical differences among three or more groups. Statistical significance was at the p < 0.05 level.

3. Results

3.1. Inhibitory Effect of Phloretin on FASN Activities and Some Partial Reactions of FASN

The overall activity of FASN was assessed in vitro to evaluate the inhibitory potential of phloretin. The results demonstrated that phloretin possessed a dose-dependent inhibitory function on FASN overall reaction activity with an IC50 value of 4.90 ± 0.66 μM, as shown in Figure B.

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Inhibitory effect of phloretin on the overall reaction of FASN. (A) The chemical structure of phloretin. (B) Relative FASN activities in the presence of phloretin at concentrations of 2.5, 5, 7.5, 10, and 12.5 μM. Data are expressed as mean ± SD (n = 3).

Phloretin’s effects on partial reactions of FASN were also examined. At 10.0 μM, phloretin inhibited 81.0% of the overall FASN activity. However, at this same concentration, only 3.5% of the β-ketoacyl reduction activity and 4.8% of the enoyl reduction activity were suppressed, indicating minimal inhibition of the KR and ER domains (Figure ). When NADPH was present in the reaction mixture, FASN could catalyze the reaction with AcAc-CoA as a substrate. The reaction entailed four domains of FASN, including MAT, KR, DH, and ER, as well as ACP in FASN. From the experimental results, phloretin almost did not affect the AcAc-CoA-involved reaction. After addition of 10.0 μM phloretin in the reaction mixture, only 19.3% activity of the overall FASN reaction remained, whereas 91.2% of the AcAc-CoA reaction activity remained (Figure ). These findings suggest that phloretin does not significantly inhibit the domains responsible for the AcAc-CoA reaction.

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Effects of phloretin (10.0 μM) on the β-ketoacyl reduction, enoyl reduction, AcAc-CoA reaction, as well as the overall reaction activity. The concentration of phloretin in the reaction system was 10.0 μM. Data are expressed as mean ± SD from three independent experiments. OA means the overall reaction; KR means the β-ketoacyl reduction; and ER means the enoyl reduction.

3.2. Irreversible Inhibitory Effect of Phloretin on FASN

Phloretin also exhibited a time-dependent inhibition of FASN, as shown in Figure . With a phloretin concentration of 2 mg/mL, the k obs value was 0.0264 min–1 for the overall reaction activity of FASN. In contrast, no time-dependent inhibition was observed for the KR domain (Figure ). This discrepancy suggests that the irreversible inactivation of FASN by phloretin is unlikely to involve the KR domain.

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Time-dependent inhibitory effect of phloretin on the overall FASN activity. FASN was incubated with 2 mg/mL phloretin, and aliquots were sampled at specified time intervals to assess the residual activity. R.A. means the relative activity.

3.3. Kinetic Mechanism of FASN Inhibition by Phloretin

Kinetic analyses were conducted to elucidate the mechanism by which phloretin inhibits FASN. By keeping the concentration of phloretin at a constant value and measuring the effect of increasing one substrate concentration (the other two substrates were fixed at a certain value) on the initial reaction rate, the possible interference of phloretin on each substrate binding site was investigated. Three families of straight lines which had different intersections were yielded by double-reciprocal plots (Figure A–C). For the substrate Ac-CoA, the lines obtained from the double reciprocal plot had an intersection on the Y axis (Figure A). The common intercept indicated that phloretin inhibited FASN in a competitive manner with Ac-CoA. The dissociation constant (K is) was calculated to be 3.46 μM according to the secondary plot. For the substrate Mal-CoA, the lines had an intersection point in the second quadrant (Figure B), which indicated that the inhibition manner against Mal-CoA was mixed competitive and noncompetitive. The dissociation constant (K is) and the dissociation constant for phloretin and the enzyme–substrate complex (K ii) were calculated to be 5.69 and 9.41 μM, respectively. However, for the substrate NADPH (Figure C), the double reciprocal plots showed a group of parallel lines, which indicated that phloretin was an uncompetitive inhibitor of FASN against NADPH. The dissociation constant was calculated from intercepts versus phloretin concentration (K ii = 1.80 μM). The inhibition types and constants of phloretin on each substrate of FASN are summarized in Table .

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Lineweaver–Burk plots illustrate the inhibitory effects of phloretin on FASN activity. (A) Concentration of Mal-CoA was 10 μM, and the concentration of NADPH was 35 μM. Ac-CoA was the variable substrate. The concentrations of phloretin were 0 μM (black circles), 1.60 μM (black diamonds), 3.42 μM (black triangles), and 5.86 μM (black squares). (B) Concentration of Ac-CoA was 3 μM, and the concentration of NADPH was 35 μM. Mal-CoA was the variable substrate. The concentrations of phloretin were 0 μM (black circles), 1.14 μM (black diamonds), 2.28 μM (black triangles), and 6.88 μM (black squares). (C) Concentration of Ac-CoA was 3 μM, and the concentration of Mal-CoA was 10 μM. NADPH was the variable substrate. The concentrations of phloretin were 0 μM (black circles), 4.56 μM (black diamonds), 6.88 μM (black triangles), and 9.12 μM (black squares). Each data point represents the mean from 2 to 5 experiments.

1. Inhibition Types for Phloretin Against Every Substrate of FASN.

Substrate Inhibitory manner Inhibition constant (μM)
K is K ii
Ac-CoA Competitive 3.46 /
Mal-CoA Mixed competitive and noncompetitive 5.69 9.41
NADPH Uncompetitive / 1.80
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In addition, the substrate protective effect of phloretin on time-dependent FASN inhibition was examined by measuring FASN inactivation by phloretin with Ac-CoA (10 μM), Mal-CoA (10 μM), or NADPH (10 μM). The inactivation conditions were the same but without the control of any substrate. The k obs values were 0.025 min–1, 0.067 min–1, 0.064 min–1, and 0.070 min–1, respectively. By comparing the constants, we found that k obs was only decreased by about 5–10% by preincubation with Mal-CoA or NADPH. These results demonstrated that the substrates of both Mal-CoA and NADPH did not show evident protective effect. However, the addition of Ac-CoA alleviated its inactivation. The results indicated that the time-dependent irreversible inactivation was related to the Ac-CoA binding sites in KS and/or MAT domains. Before addition of phloretin, FASN of different concentrations was premixed with the substrate to further analyze the protective effect of Ac-CoA. k obs decreased with the increase of substrate concentration. A linear relationship between 1/k obs and the Ac-CoA concentration was obtained (Figure ), indicating that Ac-CoA could competitively protect FASN against phloretin.

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Substrate protection by Ac-CoA against the phloretin-induced inhibition of FASN. FASN (0.60 μM) was preincubated with 6.6 μM phloretin in the presence of increasing concentrations of Ac-CoA. A linear inverse relationship between 1/k obs and Ac-CoA concentration was observed.

3.4. Inhibition of Phloretin on KS Domain of FASN

The docking results revealed that phloretin-KS exhibited a binding energy of −7.86 kcal/mol, while C75-KS showed −6.83 kcal/mol, indicating that both compounds could stably bind to KS. The lower binding energy of phloretin suggests its higher binding affinity toward KS. Their identical binding sites implied a similar mechanism of action. As shown in Figure , phloretin formed five hydrogen bonds with KS, involving residues Thr 262, His 293, Thr 295, and Asp 301, whereas C75 established three hydrogen bonds with residues Asp 254, Thr 262, and Gln 269. The significantly greater number of hydrogen bonds formed by phloretin compared with C75 demonstrates a more stable binding interaction between phloretin and KS.

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Molecular docking analysis of phloretin and C75 with the KS domain of FASN. (A) and (B) The interaction of phloretin and C75 with the FAS-KS domain, respectively.

To further investigate the binding stability of small molecules with the protein, we performed molecular dynamics simulations (MDSs). As shown in Figure , a 100 ns MDS was conducted to collect comprehensive binding information. Root mean square deviation (RMSD) analysis revealed that the phloretin–KS complex exhibited smaller conformational fluctuations and reached a stable state faster compared to the C75–KS complex. The RMSD value of phloretin remained largely within 0.4 nm throughout the simulation, while C75 stabilized after 50 ns with an RMSD value of around 0.5 nm. These results indicate that both phloretin and C75 maintained stable binding with KS, with phloretin adopting a more stable binding conformation. Radius of gyration (Rg) analysis demonstrated that KS retained its compactness and structural stability during the 100 ns simulation, showing no significant global contraction or expansion. Root mean square fluctuation (RMSF) results suggested that phloretin and C75 induced similar conformational effects on the protein. Residues 120–130 and 252–262 exhibited higher flexibility, with residue 262 located near the ligand-binding site, implying that ligand binding may enhance local fluctuations in these regions. These flexible regions are likely associated with potential active sites for ligand–receptor interactions. Hydrogen bonding, a critical stabilizing force between ligands and receptors, directly reflects binding stability. Both phloretin and C75 maintained stable hydrogen bonds with KS during the simulation. Notably, phloretin formed significantly more hydrogen bonds than C75 after 50 ns, further confirming its superior binding stability. Collectively, these molecular dynamics simulations validated the effective and stable binding of phloretin to KS.

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MDS results of phloretin and C75 with the KS domain of FASN. (A and B) RMSD analysis reveals that the phloretin–KS complex exhibits lower structural fluctuations and reaches equilibrium faster than the C75–KS complex during molecular dynamics simulations. (C) The stable Rg values over the 100 ns simulation indicate that KS maintains its compactness and structural integrity without significant global contraction or expansion. (D) RMSF profiles demonstrate comparable conformational effects on the protein structure upon binding of phloretin and C75. (E) Both phloretin and C75 form stable hydrogen bonds with KS throughout the simulation. Notably, phloretin maintains a significantly higher number of hydrogen bonds than C75 after 50 ns.

3.5. Inhibitory Effects of Phloretin on Cell Viability, FASN Expression, Intracellular FASN Activity, and the Intracellular TG Content in Breast Cancer Cells

To evaluate the effects of phloretin on intracellular FASN, we measured its impact on cell viability, FASN expression levels, and FASN activity in MDA-MB-231 and MCF-7 cancer cells. After incubating the cells with varying concentrations of phloretin for 24 h, cell viability assays revealed IC50 values of 81.4 μM for MDA-MB-231 and 101.1 μM for MCF-7 (as shown in Figure ), indicating that phloretin effectively inhibits cancer cell growth and reduces survival rates. Furthermore, to determine whether phloretin suppresses FASN expression, we analyzed FASN expression levels following 24 h of treatment with different drug concentrations. The results demonstrated that phloretin at concentrations ranging from 60–90 μM significantly decreased FASN protein content in both cell lines (Figure ).

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Effects of phloretin on viability and FASN expression in breast cancer cells. (A and D) Cell viabilities were measured by spectrophotometrically monitoring oxidation of NADPH at 340 nm. (B and E) Expression levels of FASN in phloretin-treated cells. (C and F) Bar graphs derived from the quantitative densitometric analysis of Western blot images. Data were expressed as means ± SD (n = 3). **p < 0.01 compared to control (0 μM); ***p < 0.001 compared to control (0 μM).

As demonstrated in Figure , phloretin concentration dependently suppressed intracellular FASN enzymatic activity in breast cancer cells. Significant inhibition (p < 0.01) was observed at concentrations as low as 30 μM, with maximal inhibition exceeding 50% at the highest tested dose (90 μM). Concomitantly, cellular TG levels exhibited a marked concentration-dependent reduction (p < 0.001). This parallel attenuation of FASN function and lipid accumulation establishes a direct mechanistic link between phloretin’s target engagement and its metabolic consequences.

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Effects of phloretin on intracellular FASN activity and TG content in breast cancer cells. Relative FASN activities were measured by spectrophotometric monitoring of the oxidation of NADPH at 340 nm in MDA-MB-231 cells (A) and MCF-7 cells (B). Data were expressed as means ± SD (n = 3). **p < 0.01 compared to control; ***p < 0.001 compared to control (0 μM). Relative TG values were measured by spectrophotometrically monitoring the oxidation of NADPH at 500 nm in MDA-MB-231 cells (C) and MCF-7 cells (D). ***p < 0.001 compared to control (0 μM).

4. Discussion

The morbidity of both obesity and cancer has increased rapidly in the past 30 years. FASN, as an indispensable enzyme regulating fatty acid metabolism in the body, has been considered as a reasonable drug action target for the treatment of both obesity and cancer. Several investigational products targeting FASN are in clinical trials. However, no FASN inhibitors have been successfully developed into drugs yet. Therefore, it is necessary to search for safe and effective FASN inhibitors in order to obtain practical application in the treatment of obesity and cancer. We selected phloretin for this study based on its previously reported lipid-lowering and anticancer properties. Prior studies have shown that phloretin suppresses hepatic steatosis, enhances insulin sensitivity, and inhibits the proliferation of various cancer cells. Moreover, its polyphenolic nature and structural similarity to other known FASN inhibitors supported its candidacy for investigation. Through screening and enzyme activity determination, we proved that phloretin is a novel and potent FASN inhibitor with stronger inhibitory ability than those of cerulenin (IC50 = 89 μM) and EGCG (IC50 = 52 μM in reference and 50.50 ± 0.75 μM in test results).

Our findings position phloretin as a mechanistically distinct and pharmacologically promising FASN inhibitor, with implications spanning metabolic syndrome and cancer therapeutics. The dose-dependent inhibition of FASN enzymatic activity (IC50: 4.90 ± 0.66 μM) aligns with prior observations of phloretin’s bioactivity in lipid-lowering models, yet the detailed kinetic and structural insights reported here significantly advance our understanding of its mode of action. In order to understand the inhibitory mechanism of phloretin on FASN, the kinetic parameters of FASN were measured. Phloretin showed strong inhibition of the FASN overall reaction. However, the inhibitory effects of phloretin on the ER, KR domain, and AcAc-CoA involved reaction were all very weak. These results indicated that ER, KR, MAT, DH, and ACP were not supposed to be the main active sites in FASN that are inactivated by phloretin, which were consistent with the kinetic results that phloretin acts on FASN in an uncompetitive manner with respect to NADPH. Lineweaver–Burk analysis of the kinetic data showed that phloretin inhibited the overall reaction of FASN in a competitive manner with respect to Ac-CoA and a noncompetitive manner with respect to Mal-CoA, which suggested that phloretin might act on the acetyl binding group but not on the malonyl group of FASN. Hence, the acting sites of phloretin on FASN were most likely on KS, but not on ER, KR, MAT, DH, TE, and ACP. Among all six domains of FASN, KS catalyzes the condensation reaction. It has been found that the main action site of several polyphenolic FASN inhibitors on the enzyme is KS. To support these biochemical results, docking simulations revealed strong and stable binding of phloretin to the KS domain, with more extensive hydrogen bonding than classical inhibitors, such as C75. Docking simulations indicated that phloretin binds favorably in the active site of FASN. The binding modes were analyzed as described above. Furthermore, our 100 ns MD simulation of the FASN–phloretin complex confirmed the stable binding of phloretin in the active site, supporting the docking predictions. These findings are consistent with literature reports that docking-predicted binding affinities correlate with actual inhibitory potency. Consistent with the results of the enzyme activity assay, by AutoDock and molecular dynamics analysis, we found that the binding ability of phloretin to KS is stronger than that of the known FASN inhibitors C75 and cerulenin. A scheme of the mechanism of phloretin that inhibits FASN is shown in Figure .

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Mechanism of phloretin acts on the KS domain of FASN.

As a plant-derived polyphenolic FASN inhibitor, EGCG acts mainly on the KR domain by binding to the same active site competitively with the substrate NADPH. C75 and cerulenin, two well-known FASN inhibitors, could inhibit FASN by irreversibly binding to the active site related to the substrate Mal-CoA on the KS domain of FASN. Unlike these inhibitors, phloretin inhibited FASN in a mixed manner (competitive and noncompetitive) with respect to Mal-CoA. Therefore, phloretin is completely different from other classical FASN inhibitors in the inhibition mechanism. Phloretin possibly affected the KS domains of FASN because it could bind to the pocket of KS with relatively low binding energies.

Although several polyphenols with FASN inhibitory effects were reported before, few time-dependent irreversible inhibitory effects on FASN were found in polyphenolic compounds. In this study, we discovered the slow-binding inhibitory effects of phloretin on FASN. Because FASN activity is essential for the growth and survival of adipocytes and cancer cells, it is not vital for normal cells. Inhibition of FASN could selectively block the growth and proliferation of these cells. Many studies have shown that FASN inhibitors have antiobesity and anticancer potential in human cancer cells and adipocytes. Cerulenin, the first discovered FASN inhibitor, inhibited adipocyte differentiation and reduced lipid accumulation. C75 reduced both body weight and food intake in obese mice. Alpha-mangostin, a natural FASN inhibitor, could prevent preadipocyte differentiation and lipid accumulation. ,,

Cellular validation in breast cancer models strengthens the translational relevance of these biochemical findings. Phloretin’s dual suppression of FASN expression and enzymatic activity correlates with significant reductions in intracellular TG accumulationa hallmark of FASN-driven lipogenesisand cancer cell viability. These effects resonate with emerging paradigms linking FASN overexpression to cancer cell survival via membrane biosynthesis, energy storage, and signaling lipid production. The concurrent inhibition of both FASN activity (direct enzymatic blockade) and expression (potential transcriptional or post-translational regulation) suggests a pleiotropic mechanism that warrants further investigation, particularly regarding phloretin’s interplay with oncogenic signaling pathways, such as PI3K/AKT or SREBP-1.

From a therapeutic perspective, phloretin’s natural origin and multitarget capacity present both opportunities and challenges. Its ability to simultaneously curb lipogenesis and proliferation aligns with the pathophysiology of obesity-associated cancers, where FASN serves as a metabolic nexus. However, the irreversibility of FASN inhibition raises questions about potential off-target effects or toxicity in normal tissues dependent on physiological lipogenesis. Future studies should evaluate phloretin’s selectivity across FASN isoforms and its impact on noncancerous adipocytes. Additionally, the compound’s pharmacokinetic propertiesincluding bioavailability, tissue distribution, and metabolismrequire systematic characterization to optimize its therapeutic window.

Phloretin, despite its natural origin and multitarget bioactivity, faces significant pharmacokinetic limitations. Pharmacokinetic analysis of phloretin in rats, administered via both oral and intravenous routes, revealed an oral bioavailability of 8.676%. Phloretin has been investigated in various rodent models, where oral doses typically range from 50 to 400 mg/kg and result in low systemic exposure due to poor bioavailability and rapid metabolism. In Sprague–Dawley rats, a single 100 mg/kg dose produced a Cmax of approximately 952 ng/mL (4.17 μM) at 15 min postdose. Higher oral doses of 200 and 300 mg/kg yielded proportional increases in AUC, but Cmax values remained below 10 μM. Disease models employing daily oral phloretin at 50–100 mg/kg demonstrated efficacy in ameliorating testosterone-induced benign prostatic hyperplasia and acetic acid-induced colitis, indicating that in vivo effective concentrations lie within the low micromolar range. , Phloretin’s poor bioavailability can be significantly improved through nanocarrier-based delivery systems, such as TPGS/Pluronic F68-modified micelles, enhancing its solubility, stability, and therapeutic potential. Phloretin’s oral bioavailability and therapeutic efficacy can be significantly enhanced through polymer-based nanoencapsulation techniques, which improve its systemic absorption, antioxidant activity, and cardioprotective effects. When formulated as an amorphous solid dispersion, phloretin shows enhanced oral bioavailability and demonstrates significant therapeutic effects in vivo, effectively attenuating NAFLD progression in mice by improving liver pathology and metabolic markers. Although phloretin has limited oral bioavailability, clinical evidence suggests that it retains biological activity when applied topically. For instance, studies have demonstrated its antiacne efficacy and its ability to protect human skin from UV-induced damage, supporting its therapeutic potential in dermatological applications. ,

Although adipocyte studies were not performed in this work, we acknowledge their relevance for the antiobesity implications of FASN inhibition. Due to the extended culture and differentiation time required to generate mature adipocytes from preadipocytes, these experiments were not included in this study but will be pursued in future work.

5. Conclusion

In summary, this study identifies phloretin as a novel FASN inhibitor with dual therapeutic potential against metabolic disorders and cancer. It uniquely and irreversibly targets the KS domain while simultaneously downregulating FASN expression, making it a compelling lead compound for next-generation therapeutics. Future research should focus on structure–activity optimization, combinatorial efficacy studies alongside current chemotherapeutic or antiobesity treatments, and rigorous in vivo validation in models relevant to disease. Given the growing global prevalence of obesity-related pathologies, phloretin’s ability to disrupt this pathogenic axis justifies expanded investigationparticularly through studies in adipocyte models to fully define its therapeutic potential.

Acknowledgments

This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA23080601); the Fundamental Research Funds for the Central Universities (Grant Nos. Y95401AXX2, E2E40604); and the Youth Innovation Promotion Association, CAS (Grant No. 2012315).

#.

T.L. and X.Z. contributed equally to this work. Conceptualization: X.M.; methodology: T.L., X.Z., X.X., C.W., and X.M.; investigation: T.L., X.Z., X.X., and C.W.; data curation: T.L., X.Z., J.X., X.X., C.W., and X.M.; writingoriginal draft preparation: T.L., X.Z., J.X., and X.M.; review and editing: J.X. and X.M.; supervision: X.M.; and funding acquisition: X.M. All authors participated in the preparation and had approved the final version of the manuscript.

The authors declare no competing financial interest.

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