Abstract Uterine fibroids, the most common benign tumors of the myometrium in women, are characterized by abnormal extracellular matrix deposition and uterine smooth muscle cell neoplasia, with high recurrence rates. Here, we investigated the potential of the marine natural product manzamine A (Manz A), which has potent anti-cancer effects, as a treatment for uterine fibroids. Manz A inhibited leiomyoma cell proliferation in vitro and in vivo by arresting cell cycle progression and inducing caspase-mediated apoptosis. We performed target prediction analysis and identified sterol o-acyltransferases (SOATs) as potential targets of Manz A. Cholesterol esterification and lipid droplet formation were reduced by Manz A, in line with reduced SOAT expression. As a downstream target of SOAT, Manz A also prevented extracellular matrix deposition by inhibiting the β-catenin/fibronectin/metalloproteinases axis and enhanced autophagy turnover. Excessive free fatty acid accumulation by SOAT inhibition led to reactive oxygen species to impair mitochondrial oxidative phosphorylation and trigger endoplasmic reticulum stress via PERK/eIF2α/CHOP signaling. The inhibitory effect of ManzA on cell proliferation was partially restored by PERK knockdown and eliminated by tauroursodeoxycholic acid, suggesting oxidative stress plays a critical role in the mechanism of action of Manz A. These findings suggest that targeting SOATs by Manz A may be a promising therapeutic approach for uterine fibroids. Keywords: Manzamine A, Uterine fibroid, SOAT, Cholesterol esterification, Oxidative stress, ER stress Graphical abstract Image 1 [37]Open in a new tab Highlights * • Sterol O-Acyltransferases (SOATs) could be a potential therapeutic marker for uterine fibroids. * • Manzamine (Manz) A inhibits cell proliferation of uterine leiomyomas through potentially targeting SOATs. * • Manz A impairs cholesterol esterification to trigger mitochondrial superoxide production and ER stress, leading to apoptosis. * • Manz A reduces extracellular matrix deposition by dismissing β-catenin-dependent transcriptional activity. Abbreviations ATF6 activating transcription factor 6 Bax Bcl-2-associated X protein Bcl-2 B-cell lymphoma 2 BiP binding immunoglobulin protein CDK cyclin-dependent kinase CE cholesterol ester CHOP C/EBP-homologous protein ECM extracellular matrix eIF2α eukaryotic initiation factor 2α ER endoplasmic reticulum FN fibronectin GAPDH glyceraldehyde 3-phosphate dehydrogenase HUtSMC human uterine smooth muscle cell IRE1 inositol-requiring enzyme 1 LC3B microtubule-associated protein 1 light chain 3 beta LD lipid droplet Manz A manzamine A MMP metalloproteinase OxPhos oxidative phosphorylation PARP poly ADP-ribose polymerase PERK protein kinase RNA-like ER kinase ROS reactive oxygen species SOAT acyl coenzyme A:cholesterol acyltransferase SQSTM1 sequestosome-1 TUDCA tauroursodeoxycholic acid UPR unfolded protein response 1. Introduction Uterine fibroids (also known as uterine leiomyomas) are the most common benign lesions of the myometrium, with an estimated incidence of 70% in women during their reproductive age [[38]1]. Uterine fibroids can be asymptomatic; however, 25% of patients with clinically apparent fibroids suffer from a reduced quality of life due to symptoms such as abnormal uterine bleeding, pelvic pain, frequent urination, infertility, and various adverse pregnancy outcomes [[39]2,[40]3]. Although the hysterectomy is the primary treatment, over 50% of patients face recurrence within five years post-operatively [[41]4]. Various treatment options for fibroids beyond a hysterectomy only provide symptomatic relief. Healthcare expenses place significant economic burdens on society and women who suffer from fibroids [[42]5]. Therefore, there is an unmet need to develop alternative medical therapeutic options for fibroids management [[43]6]. The features of leiomyomas have been well-studied, but their etiology remains largely unclear. Leiomyomas are characterized by an increased and disorganized proliferation of smooth muscle cells with abnormal deposition of extracellular matrix (ECM) [[44]7]. In addition, genetic abnormalities and signaling pathways that arise from steroid hormones and growth factors contribute to the pathogenic process of uterine fibroids [[45][8], [46][9], [47][10]]. However, effective medical treatment to eliminate fibroids is unavailable due to a lack of knowledge of their pathophysiology. Several natural products were reported to possess antitumor potential against uterine fibroids, such as vitamin D [[48]11,[49]12], resveratrol [[50]13,[51]14], epigallocatechin gallate [[52]15], and fucoidan [[53]16], due to their bioactive features, such as anti-oxidation, proliferation inhibition, or ECM protein downregulation. Thanks to the chemical and biological diversity of the oceans, several marine-derived substances are currently undergoing study to discover new anti-cancer drugs [[54]17]. Manzamine (Manz) A is a β-carboline alkaloid isolated from several marine sponge species and was initially described as a potent antitumor lead [[55]18] whose activity has been proven in pancreatic adenocarcinoma and colorectal cancer. Manz A sensitized pancreatic adenocarcinoma cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis and worked as a potential uncoupler of vacuolar ATPases to block autophagic flux and cause cell death in pancreatic adenocarcinoma cells [[56]19,[57]20]. Our previous study examined the anticancer activity and mechanism of action of Manz A in colorectal carcinoma (CRC) cells. We performed a transcriptomics analysis to reveal that Manz A induced cell cycle arrest, triggered apoptotic cell death, and abrogated the epithelial-to-mesenchymal (EMT) transition process in CRC cells, implying Manz A is a potential therapeutic agent to remodel ECM [[58]21]. In the present study, we hypothesize that uterine fibroids (leiomyomas) might benefit from the ECM remodeling activity of ManzA. We determined the effect of Manz A in Eker rat ELT-3 cells, a well-established uterine leiomyoma model, and showed that Manz A diminished cell proliferation in vitro and in vivo by producing reactive oxygen species (ROS). Acyl coenzyme A : cholesterol acyltransferase (SOAT) was predicted to be a potential target of Manz A. Manz A inhibited SOAT2 expression and lipid droplet contents, subsequently diminishing the expression of ECM-associated protein and matrix metalloproteinases (MMPs) through β-catenin signaling. Excessive ROS caused endoplasmic reticular (ER) stress response and mitochondrial oxidative stress. 2. Materials and methods 2.1. Reagents and antibodies Manz A, with a purity of >98%, was obtained from Enzo Life Sciences (Farmingdale, NY, USA). The SOAT inhibitor, avasimibe, was acquired from Cayman Chemical (Ann Arbor, MI, USA). Tauroursodeoxycholic acid (TUDCA) was purchased from MedChemexpress LLC (Middlesex, NJ, USA). DNase-free RNase A, propidium iodide (PI), Triton X-100, and Giemsa stain were purchased from Sigma-Aldrich. FITC Annexin V Apoptosis Detection Kit I was purchased from BD Biosciences (San Jose, CA, USA). Trypan blue, Halt™ Protease and Phosphatase Inhibitor Cocktail, trypsin-EDTA, TRIzol™ Reagent, RevertAid RT Reverse Transcription Kit, Power SYBR Green Master Mix, carboxy-H[2]DCFDA, and Lipofectamine 3000 were purchased from Thermo Fisher Scientific (Boston, MA, USA). The Direct-zol RNA MiniPrep kit was purchased from Zymo Research (Orange, CA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation (MTS) assay was purchased from Promega (Madison, WI, USA). The bicinchoninic acid (BCA) assay kit was purchased from T-Pro Biotechnology (New Taipei City, Taiwan). The Western Bright ECL chemiluminescent horseradish peroxidase (HRP) substrate was purchased from Advansta (Menlo Park, CA, USA). Antibodies against β-catenin, phospho-p53 (Ser15), phospho-eIF2α (Ser51), eIF2α, PERK, BiP, C/EBP-homologous protein (CHOP), Bcl-2, Bax, caspase-3, poly ADP-ribose polymerase (PARP), and LC3B were purchased from Cell Signaling Technology (Beverley, MA, USA). Antibodies against SOAT2, sequestosome-1 (SQSTM1)/p62, and lamin B1 were purchased from GeneTex (Irvine, CA, USA). An antibody against GAPDH was purchased from Proteintech (Chicago, IL, USA). Antibodies against α-tubulin and actin were purchased from EMD Millipore (Billerica, MA, USA). An antibody against activating transcription factor 6α (ATF-6α) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Antibodies against cyclin-dependent kinase 2 (CDK2), CDK4, and oxidative phosphorylation (OxPhos), and the goat anti-rabbit/mouse antibody immunoglobulin G (IgG) were purchased from Abcam (Cambridge, UK). 2.2. Cell culture The Eker rat-derived uterine leiomyoma cell line Eker Leiomyoma Tumor-3 (ELT-3) [[59]22] (American Type Culture Collection, Manassas, VA, USA) and primary human uterine smooth muscle cells (HUtSMCs) (PromoCell, Heidelberg, Germany) were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere containing 95% air and 5% CO[2] at 37 °C. 2.3. In vivo animal experiments All the animal studies were conducted according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Taipei Medical University (LAC-2022-0058). Five-week-old female BALB/c mice (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan) were housed under a 12 h light/12 h dark cycle in a pathogen-free environment, with ad libitum access to food and water. ELT-3 cells (1.5 × 10^5) were sealed in MTAM (Bioman Scientific Co., LTD) and incubated in a complete medium prior to transplantation. BALB/c mice were anesthetized, and about 1–2 cm of skin incisions were made on the back using a scalpel. Cell-loaded MTAMs were then implanted subcutaneously (s.c.) using a micro spatula. The incisions were closed with sutures. After three days of observation, mice were assigned to two groups (n = 5 for each group), intraperitoneally injected with Manz A (2 mg/kg/day) or DMSO once daily for ten days with the body weight monitored. At the end of the experiment, the mice were euthanized. The MTAM membranes were retrieved, and the cell viabilities were assessed by MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay. Briefly, respective cell containing MTAMs were cut into small pieces and then incubated with 600 μL of 0.5% MTT solution for 3 h. The crystal formazan of each MTAM was dissolved in 300 μL dimethyl sulfoxide (DMSO) for 15 min. The absorbance was measured on an Epoch Microplate Spectrophotometer at a wavelength of 570 nm. 2.4. Cell viability assay The effects of Manz A on cell viability were assessed using an MTS assay (CellTiter 96 Aqueous One Solution cell proliferation assay; Promega, Madison, WI, USA). Briefly, cells were seeded in 96-well plates at a density of 3000 cells/well overnight. MTS reagent was added to each well after Manz A treatment for indicated incubation times and incubated at 37 °C for 90 min in the dark. The spectrophotometric absorbance of colored formazan generated by viable cells was measured on an Epoch Microplate Spectrophotometer (BioTek, Winooski, VT, USA) at 490 nm. The relative cell viability was defined as the ratio of the absorbance of Manz A-treated cells to that of DMSO-treated ones. 2.5. Colony formation assay The colony formation assay was performed to evaluate the long-term inhibitory effects of Manz A on cell proliferation. ELT-3 cells were seeded in 6-well plates at a density of 2.5 × 10^4 cells/well. After attachment, cells were treated with 2.5 or 5 μM Manz A or 0.1% DMSO (vehicle control) for 48 h. Cells were then detached, reseeded in 10-cm dishes at a density of 1000 cells, and cultured in a drug-free medium for seven days to allow colonies to form. The culture medium was changed every two days to ensure sufficient nutrient supplementation. Formed colonies were fixed in methanol and stained with 1:10 Giemsa stain (Sigma-Aldrich). 2.6. Flow cytometric analysis of apoptosis and the cell cycle ELT-3 cells were seeded in 10-cm dishes at a density of 2.5 × 10^5 cells overnight for attachment and treated with 2.5, or 5 μM Manz A or 0.1% (v/v) DMSO vehicle control for a further 24 h. Cells were then washed with PBS and harvested by trypsin. For the cell cycle analysis, cells were fixed with 70% (v/v) ethanol at −20 °C overnight, washed with ice-cold PBS, incubated with 0.1 mg/mL DNase-free RNase A for 30 min, and stained with 50 μg/mL propidium iodide (PI) for a further 30 min. The DNA content of PI-stained single-cell suspensions was acquired with a BD Accuri C6 cytometer (BD Biosciences, Franklin, NJ, USA). A FITC Annexin V Apoptosis Detection Kit I (BD Biosciences) was used for the apoptosis assay. Cells were suspended with 100 μl of binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, and 2.5 mM CaCl[2] at pH 7.4) and stained with 2 μl of FITC-conjugated Annexin V and 2 μl of PI (50 μg/mL) for 15 min at room temperature (RT) in the dark and analyzed with a BD Accuri C6 cytometer (BD Biosciences). 2.7. Detection of the mitochondrial membrane potential (MMP) The MMP was evaluated with JC-1 (Thermo Fisher Scientific) staining. JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼529 nm) to red (∼590 nm). Consequently, mitochondrial depolarization can be indicated by a decrease in the red/green fluorescence intensity ratio [[60]23]. ELT-3 cells treated with Manz A or vehicle control were harvested and stained with 2 μM JC-1 dye in PBS for 30 min in the dark at 37 °C. The MMP was assessed by an Attune™ NxT Flow Cytometer using the BL1 channel with Ex/Em of 515/529 nm and BL3 channel with Ex/Em of 585/590. The red-to-green fluorescence ratio was calculated to infer the MMP alterations. 2.8. Detection of intracellular reactive oxygen species (ROS) Cellular ROS levels were evaluated with the cell-permeant probe 6-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H[2]DCFDA; Thermo Fisher Scientific). Briefly, cells seeded in 6-well plates at the density of 8 × 10^4 overnight were pretreated with or without 5 mM N-acetylcysteine (NAC) for 1 h before Manz A treatment for a further 16 h. After washing once with PBS, cells were incubated with 20 μM carboxy-H[2]DCFDA in serum-free DMEM at 37 °C and 5% CO[2] in the dark for 45 min. Stained cells were washed once with PBS to remove any remaining dye. For the hydrogen peroxide (H[2]O[2])-treated positive control, cells were incubated in DMEM with reduced serum (1%, v/v) containing 1 mM H[2]O[2] for 30 min. Fluorescent images were taken with a Leica DM IL inverted fluorescence microscope (Leica Microsystems, Wetzlar, Germany). For the flow cytometric analysis, treated cells were harvested and stained with carboxy-H[2]DCFDA. ROS levels were measured using an Attune™ NxT Flow Cytometer (Thermo Fisher Scientific) by the BL1 channel. Mitochondrial superoxide was assessed with MitoSOX™ Red (Thermo Fisher Scientific). Cells treated with Manz A or DMSO were harvested and incubated with 5 μM MitoSOX in PBS for 25 min in the dark at 37 °C. For the positive control, cells were stained with MitoSOX for 10 min and treated with 10 μM antimycin A/rotenone with continuous incubation of MitoSOX for a further 15 min. ROS levels were assessed by an Attune™ NxT Flow Cytometer (Thermo Fisher Scientific) using the BL2 channel. 2.9. Gelatin substrate gel zymography Gelatinolytic activities of MMP-2 and MMP-9 were performed using zymography as described previously [[61]24] with modifications. In the gel zymographic assay, gelatin is incorporated into sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and can be degraded by active metalloproteases, such as MMP-2 and MMP-9, resulting in negative (unstained) bands. This assay can detect active and latent gelatin bands on gels based on their molecular weight. Briefly, ELT-3 cells were seeded into 6-well plates at a density of 3 × 10^5 cells/well for 24 h. Cells were treated with Manz A or DMSO in serum-free DMEM for 48 h. Subsequently, 2 mL of each clarified conditioned medium was collected and centrifuged at 400×g for 10 min to remove cells and debris. The protein concentration was determined by a BCA protein assay. The clarified supernatant (10 μg of total protein) was mixed with 5 × SDS sample buffer (250 mM Tris-HCl at pH 6.8, 40% glycerol (v/v), 8% SDS (w/v), 0.01% bromophenol blue (w/v)) containing no reducing agent, and it was then subjected to SDS-PAGE in a 7.5% SDS-PAGE gel containing 0.1% gelatin (v/v, Sigma-Aldrich). After electrophoresis, gels were incubated with a renaturing solution (2.5% Triton X-100 (v/v)) for 60 min (3 times at 20 min each) at RT with gentle agitation to remove SDS. Gels were incubated at 37 °C in incubation buffer (50 mM Tris-HCl, 5 mM CaCl[2], and 0.02% NaN[3] (w/v) at pH 7.6) for 16 h, stained with 0.5% Coomassie Brilliant Blue R250 (w/v) in 30% methanol (v/v) and 10% acetic acid (v/v) for 30 min, and then destained in 10% acetic acid (v/v) and 30% methanol (v/v) to clearly visualize the digested bands. Proteolytic activities of MMP-2 and MMP-9 were visualized as clear bands against the blue background of stained gelatin. 2.10. Cholesterol ester (CE) measurement The concentration of CE was measured with a Total Cholesterol and Cholesteryl Ester Colorimetric/Fluorometric Assay Kit (BioVision, Mountain View, CA, USA). In brief, 2 × 10^6 ELT-3 cells with indicated treatment were centrifuged, and the pellet was re-suspended in 400 μl of a chloroform : isopropanol : NP-40 (7 : 11 : 0.1) solution using a micro-homogenizer. The organic phase was transferred to a new tube after being centrifuged at 15,000 × g for 10 min and then air-dried at 65 °C. Dried lipids were re-dissolved in 200 μl of Assay Buffer with vortexing until homogeneous. The reaction was conducted following the instruction manual. The optical density (OD) was measured with a spectrophotometer at a wavelength of 570 nm. 2.11. Bodipy 493/503 and MitoTracker staining Cells were seeded on coverslips and treated with 5 μM Manz A or DMSO for 24 h. Cells were stained with 100 nM of MitoTracker™ Red CMXRos (Thermo Fisher Scientific) for 45 min in the dark. Following three washes of PBS, cells were fixed with 4% paraformaldehyde/PBS (w/v) for 15 min and stained with Bodipy 493/503 (Cayman Chemical) for 30 min at RT in the dark. EverBrite™ Mounting Medium with 4′,6-diamidino-2-phenylindole (DAPI; Biotium, Fremont, CA, USA) was used to immobilize the coverslips. Coverslips were sealed with nail polish. Fluorescent images were captured using a Leica DM IL inverted fluorescence microscope. 2.12. Immunocytochemistry ELT-3 cells were cultured on sterile glass coverslips and treated with 5 μM Manz A or DMSO for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.1% Triton X-100/PBS (v/v) for 10 min. Cells were then blocked in BlockPRO blocking buffer (Visual Protein, Taipei, Taiwan) at RT for 1 h and incubated with primary antibodies overnight at 4 °C. After being washed three times with Tris-buffered saline containing 0.1% Tween-20/TBS (v/v, TBST), cells were further incubated with secondary antibodies (Alexa Fluor® 488 goat anti-rabbit IgG and Alexa Fluor® 546 goat anti-mouse IgG, Thermo Fisher Scientific) for 1 h at RT in the dark. After several washes with TBST, cells were mounted with EverBrite™ Mounting Medium with DAPI. Fluorescent images were taken with a Leica DM IL inverted fluorescence microscope or a Leica TCS SP5 confocal spectral microscope. 2.13. PERK knockdown and transfection To silence expression of PERK, small interfering (si)RNA for PERK was used. ELT-3 cells were seeded in 6-well plates at 50%–70% confluence and transfected with 25 nM PERK siRNA (r) (sc-60074) or control siRNA (sc-37007, Santa Cruz Biotechnology, Santa Cruz, CA, USA) using Lipofectamine 3000. After 24 h of transfection, transfected cells were subsequently treated with 5 μM Manz A or DMSO for 24 h. Protein expression of PERK and downstream signaling was confirmed by western blot. 2.14. Real-time quantitative polymerase chain reaction (qPCR) analysis Total RNA from ELT-3 cells was extracted using TRIzol™ Reagent (Thermo Fisher Scientific) followed by purification with Direct-zol RNA MiniPrep (Zymo Research, Irvine, CA, USA). Complementary DNA (cDNA) was reverse-transcribed from 2 μg total RNA using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific). Messenger RNA (mRNA) levels of genes of interest were measured with gene-specific primers (Fn1: forward, GGCCAGTCCTACAACCAGTAT and reverse, TCGGGAATCTTCTCTGTCAGC; GAPDH: forward, TGCACCACCAACTGCTTAGC and reverse, GGCATGGACTGTGGTCATGAG) and Power SYBR Green Master Mix (Thermo Fisher Scientific). Briefly, 1 ng of cDNA was added to the mix containing appropriate primer sets (400 nM) and SYBR green in a 10-μL reaction volume. All samples were analyzed in triplicate. Real-time PCR analyses were performed using an Applied Biosystems StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific). Amplification was performed under the following cycling conditions: denaturation at 95 °C for 10 min followed by 40 cycles of 15 s at 95 °C and 30 s at 60 °C. Synthesis of the DNA product of the expected size was confirmed by a melting curve analysis and DNA electrophoresis. Relative quantification was done with the ΔΔCt method, which normalized to the internal control GAPDH and vehicle control. 2.15. Isolation of nuclear and cytoplasmic proteins Cells were washed with ice-cold PBS and harvested. Cells were then homogenized with hypotonic buffer (10 mM Tris-HCl at pH 7.4 and 0.5 mM MgCl[2]) containing PPI by 20 strokes with a 22-G needle. Nuclear fractions were pelleted by centrifugation at 2500×g for 10 min at 4 °C and washed with ice-cold PBS. Supernatants containing cytosol and other fractions were collected. Western blot analysis of α-tubulin and lamin B1 indicated no contamination between the nuclear and cytoplasmic fractions. 2.16. Western blot analysis ELT-3 cells treated with or without Manz A for the indicated times were lysed in ice-cold RIPA buffer (20 mM Tris at pH 7.5, 300 mM NaCl, 2% SDC (w/v), and 2% NP40 (v/v)) containing PPI. The protein concentration was determined by a BCA protein assay. Proteins at 20 μg were subjected to SDS-PAGE, resolved on a 7.5%–15% polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane. Membranes were then blocked in BlockPRO™ Blocking Buffer (Visual protein) for 1 h at RT and incubated with the appropriate primary antibody overnight at 4 °C. After three washes with TBST, the membrane was incubated with secondary anti-rabbit or anti-mouse IgG antibodies for 1 h at RT. The immunoreaction was visualized using the enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate and detected using a Luminescent Image Analyzer Amersham Imager 600 (GE Healthcare Life Sciences, Little Chalfont, UK). The band intensity was quantified using ImageJ software. 2.17. Quantitative proteome analysis ELT-3 cells were seeded in 6 cm dishes overnight and treated with Manz A (5 μM) or 0.1% DMSO as a control for 24 h in biological triplicate. Cells were lysed in lysis buffer (12 mM SDC, 12 mM SLS, 0.1 M TEAB or Tris-HCl (pH 9.0), 5 mM EDTA) containing PPI and sonicated with 60% amplitude and 0.6 cycle using ultrasonic homogenizers LABSONIC M (Sartorius, Goettingen, Germany) for 2 min on ice. Protein extracts were clarified by centrifugation at 17,000 × g for 20 min at 4 °C. Protein concentration was determined using BCA protein assay and stored at −80 °C until further analysis. The protein lysate was subjected to reduction using 10 mmol/L Tris (2-carboxyethyl) phosphine at 37 °C for 30 min, followed by alkylation with 25 mmol/L chloroacetamide for 30 min at 37 °C. Subsequently, the protein solution was digested using endoproteinase Lys-C (1/100 w/w) at RT for 3 h, and then with sequencing-grade trypsin (1/100 w/w, Promega) overnight at 37 °C. To acidify the digested samples, trifluoroacetic acid (TFA) was added to achieve a pH of 2, and an equal volume of ethyl acetate (EtOAc) was mixed. After centrifugation at 17000 ×g for 2 min at RT to remove detergent completely, the samples were dried with a centrifugal vacuum evaporator. Dried peptides were reconstituted in 0.1% (v/v) TFA, and the pH was adjusted to a range of pH 2–3 and desalted using StageTips with SDB-XC Empore membrane (3 M Company). Peptide samples were quantified at OD214 and equal amount of peptide from each sample was dried with a centrifugal evaporator. Peptides were dissolved with 200 mM HEPES and labeled with TMT 18-plex™ isobaric reagents (Thermo Fisher Scientific) for 1 h at RT. The reaction was quenched with 0.33% hydroxylamine at RT for 15 min and acidified with the addition of TFA. The TMT-labeled samples were combined, vacuum-dried, desalted, basic reversed-phase fractionated, and desalted prior to LC/MS/MS analysis. LC/MS/MS analysis was performed on an Orbitrap Fusion Lumos Tribrid quadrupole-ion trap-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) coupled to an Ultimate system 3000 nanoLC system (Thermo Fisher Scientific, Bremen, Germany). Peptide mixtures were loaded into a C18 Acclaim PepMap NanoLC column (25 cm length, 75 μm inner diameter) (Thermo Scientific, San Jose, CA, USA) packed with 2 μm particles with a pore of 100 Å. The following LC buffers were used: mobile phase A (0.1% (v/v) formic acid in Milli-Q water) and buffer B (0.1% formic acid in 100% ACN). Peptides were separated by 2%–40% solvent B in 50 min at a flow rate of 300 nL/min. The mass spectrometer was operated in a data-dependent mode and automatically switched between MS1 and MS2 (MS/MS) acquisition. 2.18. MS data processing and bioinformatics analysis MS data was processed with Maxquant (version 2.4.0.0) [[62]25]. MS2 data were searched against Rattus norvegicus (Rat) and Mus musculus (Mouse) UniProt databases by Andromeda search engine. Trypsin/P was specified as a digested enzyme with up to two miscleavages allowed. Carbamidomethylation on cysteine was specified as a fixed modification. Methionine oxidation and N-terminal protein acetylation were specified as the variable modifications. Statistical analysis was performed using Perseus (version 2.0.3.1) [[63]26]. After filtering out the reverse hits and contaminations, values were log2 transformed and quantile normalized using NormalyzerDE [[64]27]. Missing values imputation was carried out from a normal distribution (downshift: 1.8, width: 0.3). Two sample test with an FDR threshold of 0.01 and s0 factor were applied to identify the significant differences of protein abundance. Differentially expressed proteins (DEPs) were subjected to functional enrichment analysis from Gene Ontology Biological Processes (GOBP) and WikiPahways database using Enricher [[65]28]. 2.19. Statistical analysis Data are shown as the mean ± standard deviation (SD). A two-tailed Student's t-test was used to determine the significance of differences between the Manz A and DMSO-treated groups. Statistical significance was accepted when p < 0.05. Data were collected from at least three independent experiments (n = 3). 3. Results 3.1. Manz A inhibits the proliferation of uterine leiomyoma cells in vitro and in vivo To evaluate the effect of Manz A on cell proliferation in uterine leiomyoma cells, we conducted an MTS assay on the ELT-3 cell line, the Tsc2-null proliferative smooth muscle cells isolated from Eker rat uterine leiomyomas [[66]22], expressing estrogen and progesterone receptors that respond to hormone [[67]29,[68]30]. We showed that Manz A significantly reduced cell viability in rat ELT-3 ([69]Fig. 1A) and HUtSMCs ([70]Fig. 1B) in dose- and time-dependent manners. The 50% inhibitory concentration (IC[50]) values were 4.5 ± 0.2 μM in ELT-3 cells, and 6.5 ± 0.3 μM in HUtSMCs after 48 h of treatment. To examine the long-term inhibitory effects of Manz A on cell growth, we performed a colony formation assay on Manz A-treated cells in a drug-free environment for 7 days and found that Manz A significantly decreased the number of colonies even though we removed the drug, suggesting the effect of MA on ELT-3 was irreversible ([71]Fig. 1C). In advance, we further investigated whether Manz A inhibited the cell growth of uterine fibroids in vivo. To this end, we applied a rapid drug screening system using a microtube array membrane (MTAM) [[72]31]. MTAMs are a novel microstructure that consists of one-to-one connected hollow fibers arranged in arrays, allowing the investigation of in vivo microenvironmental interactions in a short time [[73]32]. Instead of forming leiomyomas, ELT-3 cells are encapsulated within hollow fibers of the MTAM, subcutaneously implanted, and followed by drug administration. Due to the homogenously porous and ultra-thin lumen walls of hollow fibers, MTAMs possess good sensitivity towards drug delivery. After transplantation, the female BALB/c mice were intraperitoneally administered with Manz A (2 mg/kg, once daily) or vehicle control DMSO for ten days. The transplanted cell viability was significantly decreased in the Manz A-treated mice ([74]Fig. 1D), implying the translational potential of Manz A in managing uterine fibroids. Fig. 1. [75]Fig. 1 [76]Open in a new tab Manz A inhibited uterine leiomyoma in vitro and in vivo. (A) Rat ELT-3 uterine leiomyoma cells and (B) human uterine smooth muscle cells (HUtSMCs) were exposed to the indicated concentrations of Manz A for 24 or 48 h. Cell viability was assessed by an MTS assay. (C) A colony-formation assay was performed to determine the long-term effects of Manz A on the growth of ELT-3 cells. Cells were pretreated with vehicle (0.1% (v/v) DMSO) or Manz A (2.5 or 5 μM) for 48 h and left for seven days to grow in a drug-free medium. Colonies were then stained with Giemsa. (D) The in vivo effect of Manz A was assessed. ELT-3 cells (1.5 × 10^5) were encapsulated into MTAMs and subcutaneously (s.c.) implanted in female BALB/c mice. Mice were administered with Manzamine A (2 mg/kg/day) or the vehicle control DMSO group (n = 5 for each group) through intraperitoneal (i.p.) injection once daily for ten days. Cell viability (%) was measured by MTT assay and is expressed as the mean ± SD. ***p < 0.001. (E) ELT-3 cells were treated with vehicle (0.1% (v/v) DMSO) or Manz A (2.5 or 5 μM) for 24 h and then subjected to a DNA content analysis by flow cytometry. Representative histograms of the cell cycle distribution are shown in the upper panel. Cell cycle distributions from three independent experiments are quantified in the bottom panel. (F) Protein expressions of cell cycle regulators in response to Manz A treatment in ELT-3 cells were analyzed by western blot. Representative blot images and quantitation of protein levels are shown. Actin was used as an internal control. Data is expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. 3.2. Manz A induces cell cycle arrest at the G[0]/G[1] phase Previous studies demonstrated that levels of several cell cycle regulatory proteins, including E2F transcription factor 1, cyclin D1, cyclin D2, and CDK2, were elevated in uterine leiomyomas compared to normal myometrium [[77]33,[78]34], inferring that deregulation of cell cycle progression was associated with cell division and proliferation of leiomyomas. Accordingly, we investigated whether Manz A changed the cell population in cell cycle progression. We performed a flow cytometry-based analysis and revealed that Manz A significantly increased the cell population at the G[0]/G[1] and the sub-G[1] phases and simultaneously diminished it at the S phase ([79]Fig. 1E). At the protein level, Manz A markedly decreased the expressions of CDK2 and CDK4, resulting in an inactive cell cycle state blocking the entry of mitosis ([80]Fig. 1F). Our findings indicated that Manz A inhibited cell cycle progression and induced cell cycle arrest at the G[0]/G[1] phase. 3.3. Manz A triggers caspase-mediated apoptotic cell death through an intrinsic pathway Next, we examined whether Manz A induced apoptosis in ELT-3 cells by FITC-conjugated Annexin V and PI double-staining assay using flow cytometry. We found that Manz A significantly increased the cell population in the sub-G[1] phase from 3.2% (vehicle control) to 20.7% (5 μM Manz A) ([81]Fig. 2A). Moreover, Manz A markedly increased both the early apoptotic cell population (Annexin V-positive cells) from 2.1% (vehicle control) to 7.1% (5 μM Manz A) and the late apoptotic cell population (Annexin V- and PI-positive cells) from 4.7% (vehicle control) to 35.3% (5 μM Manz A) ([82]Fig. 2B). In addition, Manz A induced protein expressions of cleaved PARP, cleaved caspase-3, p-p53 (ser15), and the ratio of the proapoptotic protein Bax to the anti-apoptotic protein Bcl-2 ([83]Fig. 2C), indicating that Manz A triggered apoptotic cell death through intrinsic caspase-mediated pathways. Fig. 2. [84]Fig. 2 [85]Open in a new tab Manz A triggered caspase-dependent apoptosis in ELT-3 cells. Cells were treated with Manz A (2.5 or 5 μM) or vehicle (0.1% DMSO) for 48 h. (A) Cells were harvested and then subjected to a DNA content analysis by flow cytometry. The percentage of cells in the sub-G[1] phase of the cell cycle was quantified. (B) Cells were harvested and stained with Annexin V-FITC and propidium iodide (PI). The fluorescent signal was measured by flow cytometry. A representative result is shown in the left panel, and the statistical analysis is shown in the right panel. (C) Protein expressions of the apoptotic regulators in response to Manz A treatment in ELT-3 cells were analyzed by western blot. The cleavage form of Caspase 3 is indicated by black arrow. Representative blot images and quantitation of protein levels are shown. Actin was used as an internal control. Values are expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. 3.4. Manz A inhibits autophagy by autophagosome turnover blockade Manz A was reported to be an autophagy inhibitor that targets vacuolar ATPases and impairs cell growth of pancreatic cancer cells [[86]35]. Here, we inspected whether Manz A caused similar inhibitory effects on autophagy in ELT-3 cells. We observed that Manz A remarkably increased the protein level of an autophagosome marker phosphatidylethanolamine-conjugated LC3B (LC3B-II) ([87]Fig. S1A). An increase in LC3B-II expression indicates a distinct role in inducing autophagy and inhibiting autophagosome turnover. Simultaneously, we also found an accumulation of SQSTM1/p62, which was inversely correlated with autophagic activity ([88]Fig. S1A), confirming that Manz A inhibited the autophagosome turnover. Consistent with the results of western blot analysis, Manz A induced colocalized and accumulated expression of LC3B and p62 in ELT-3 cells ([89]Fig. S1B). 3.5. Manz A potentially targets SOAT2 and blocks cholesterol esterification and LD formation To discover the possible targets of Manz A, SwissTargetPrediction was used based on its structural similarities to known ligands. Four potential genes were predicted as Manz A targets with high probability ([90]Fig. 3A). Among them, acyl coenzyme A:cholesterol acyltransferase 1 (SOAT1) and SOAT2 were top-ranked enzymes catalyzing the formation of cholesteryl esters (CEs) from cholesterol and fatty acids (FAs). To evaluate whether the SOAT expression is correlated with leiomyoma progression, we extracted three independent public datasets from GEO. We found that SOAT2 was upregulated in fibroid tissues compared to normal myometrium ones ([91]GSE64763, [92]Fig. 3B) and associated with the MED12 G44D mutation, which has been reported as a high frequent mutation in uterine fibroids ([93]GSE128229, [94]Fig. 3C) [[95]36]. Furthermore, SOAT2 was upregulated in leiomyoma compared with the adjacent myometrium specimens collected in the proliferative phase of the menstrual cycle ([96]GSE95101, [97]Fig. 3D). These results provide clinical evidence in pathologically overexpression of SOAT2 in uterine leiomyomas. Fig. 3. [98]Fig. 3 [99]Open in a new tab Manz A targets acyl coenzyme A:cholesterol acyltransferase 2 (SOAT2), which is upregulated in uterine leiomyomas. (A) Target prediction of Manz A. Molecules with a high probability according to SwissTargetPrediction are shown. (B–D) SOAT2 was upregulated in uterine leiomyoma tissues compared to normal tissues. Relative mRNA levels of SOAT2 in 29 normal myometrial (Myo.) tissues and 25 fibroid (Fbr.) tissues from the [100]GSE64763 dataset (B), in 15 leiomyoma (MED12 G44 mutant) and 15 matched normal myometrial (wild type) patient tissue samples from the [101]GSE128229 dataset (C), and in pairs of leiomyoma and matched myometrial specimens collected at distinct phases of the menstrual cycle from the [102]GSE95101 dataset (D) are shown. (E) Western blot analyses of SOAT2 protein expression in response to Manz A treatment for 24 h in ELT-3 cells. (F) ELT-3 cells were treated with DMSO (control) or Manz A (5 or 10 μM) for 24 h, or the known SOAT inhibitor, avasimibe (Ava., 20 μM), for 48 h. Intracellular cholesterol ester (CE) levels were then determined by a cholesterol assay kit. (G) Fluorescence microscopic images show the cellular distribution of lipid droplets by Bodipy 493/503 staining. All cells were counter-stained for nuclei using DAPI (blue). Scale bar indicates 40 μm. Data is expressed as the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. SOAT is an integral membrane protein of the rough ER which forms CEs from cholesterol and FAs and controls the LD formation [[103]37]. To confirm whether Manz A targeted SOAT2, we examined SOAT2 expression, CE contents, and the lipid deposition in response to Manz A treatment. Manz A downregulated SOAT2 expression ([104]Fig. 3E), in line with the decreased contents of CEs ([105]Fig. 3F) and LDs in ELT-3 cells ([106]Fig. 3G), providing evidence that Manz A targets SOAT2 and reduces its enzyme activity. 3.6. Proteome analysis reveals Manz An upregulates the processes of ER stress and oxidative stress, and downregulates the processes of extracellular matrix assembly, DNA replication, DNA repair and cell cycle To gain a comprehensive understanding of the detailed molecular regulation of Manz A, we applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with Tandem Mass Tag (TMT)-based quantitative proteomics. A total of 4077 protein groups were identified and 3915 of them were quantifiable ([107]Fig. S2). We applied the S0 factor calculated from the Significance Analysis of Microarrays (SAM) in a two-sample t-test, with a false discovery rate (FDR) less than 1% to determine statistically significant differentially expressed proteins (DEPs). We identified 440 DEPs with 233 upregulated and 207 downregulated proteins in Manz A treatment compared with vehicle control in ELT-3 cells ([108]Fig. 4A and B). DEPs were subjected to pathway enrichment analysis form databases including gene ontology biological processes (GOBP) and WikiPahways using Enricher. The upregulated proteins were enriched in the terms of NRF2 pathway (WP2884), response to unfolded protein (GO:0006986), and cellular response to oxidative stress (GO:0034599) ([109]Fig. 5A), demonstrating that Manz An upregulated proteins participating in the processes of ER stress and oxidative stress. On the other hand, the downregulated proteins were enriched in the terms of miRNA targets in ECM and membrane receptors (WP2911), DNA replication (WP466), DNA metabolic process (GO:0006259), nucleotide excision repair (WP4753), positive regulation of extracellular matrix assembly (GO:1901203), and G1 to S cell cycle control (WP45) ([110]Fig. 5B), indicating that Manz A dysregulated the processes of extracellular matrix assembly, DNA replication, DNA repair and cell cycle. Fig. 4. [111]Fig. 4 [112]Open in a new tab Proteome analysis of Manz A effects on ELT-3 cells. (A) Volcano plot of quantified proteins constructed from log[2](fold change) (x-axis) and –log (p-value) (y-axis). Red dots indicate upregulated proteins. Blue dots indicate downregulated proteins. (B) Heatmap of differentially expressed proteins analyzed by hierarchical clustering. Each column indicates an individual sample. Each row indicates a protein. Colors represent upregulated (red) and downregulated (blue) expression between groups. (For interpretation of the references to color in this figure