Cell lines and primary tumor samples
OVTOKO and OVMANA cell lines were obtained from the Japanese Collection of Research Bioresources (Osaka, Japan), ES-2 from the American Type Culture Collection, and KK from the National Defense Medical College [10]. Primary tumor samples were obtained during surgery from 39 patients treated at Teikyo University Hospital in Tokyo, with written consent obtained from each patient after approval was approved by the Clinical Ethics Committee of the Medical Faculty at Teikyo University (approval number: 13-003-4 on 22 October 2020). The study adheres to the provisions specified in the Declaration of Helsinki.
Proliferation assay
The effects of paclitaxel on cell proliferation were evaluated using a WST-8 assay (Dojindo, Kumamoto, Japan). The assay can be used to evaluate cell viability in cell proliferation assays. WST-8 is reduced by dehydrogenase activity in cells to produce a water-soluble yellow-colored formazan dye formed by NADH produced in the mitochondria. The amount of formazan dye produced is directly proportional to the number of living cells. The protocol is described briefly as follows. Cells were seeded in 24-well plates and pre-incubated at 37°C for 24 h to enable cell attachment. At 72 h after drug treatment, 50 µL of WST-8 solution was added to each well and incubated at 37°C for 0.5–4 h (the reaction time was adjusted according to the cell line). The augmentation of enzyme activity increased the amount of formazan dye, which was quantified using a microplate reader (Bio-Rad, Tokyo, Japan) by measuring absorbance at 450–550 nm. This procedure was repeated at least three times.
Tissue immunohistochemistry and immunofluorescence
Formalin-fixed paraffin-embedded (FFPE) tissue specimens were sliced at 4 µm thickness, deparaffinized in xylene, and rehydrated using graded ethanol solutions. After activation in citric acid buffer at 98°C for 40 min, the slides were stained with anti-NNMT (Santa Cruz, Tokyo, Japan G-4; 1:100) antibodies and processed using the EnVision FLEX Visualization System (Agilent Technologies). Samples were counterstained with Mayer’s hematoxylin (131–09665, Fujifilm, Tokyo, Japan).
Tissue microarray analysis
We analyzed NNMT expression using a tissue microarray (TMA) for 39 patients with ovarian clear cell adenocarcinoma who underwent surgery at Teikyo University Hospital between January 2003 and December 2012. Detailed information about the clinical characteristics was obtained after a retrospective review of the medical records. NNMT immunohistochemical reactivity was scored without knowledge of the clinical outcomes by two observers (R.K. and Y.S.). Each sample was scored based on the percentage of positive cells in each compartment (0, no staining; 1, < 30%; 2, 30–50%; 3, ≥ 50%). The staining intensity was similar across all samples. Expression was considered ‘low’ if the cytoplasmic staining intensity was 0 or 1 and ‘high’ if scores of 2 or 3 were obtained. The analysis was limited to patients with ovarian clear cell carcinoma (N = 39). Kaplan–Meier survival curves and log-rank test were used to compare the difference and overall survival were generated using BellCurve for Excel.
Fluorescence-labeled two-dimensional differential gel electrophoresis (2D-DIGE)
Proteins were labeled using the CyDye DIGE Fluor minimal dye (GE Healthcare), as per the manufacturer’s instructions.
Briefly, 50 mg of a sample mixture extracted from the ovarian clear cell cancer cell lines (OVTOKO, OVMANA, ES-2, and KK) was adjusted to pH 8.5 using 50 mM NaOH, and samples were labeled with 400 pmol Cy5 as a control or with 400 pmol Cy3. Fluorescence labeling was performed on ice in the dark for 30 min and the reaction was subsequently quenched with 1 mL of 10 mM lysine (Sigma-Aldrich, St. Louis, USA) for 10 min. Each preparation was treated with two sample buffers containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% immobilized pH gradient (IPG)-buffer with a pH range of 4–7, and 2% dithiothreitol (DTT) according to the manufacture’s recommendations. The final volume was adjusted to 260 mL using rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% IPG buffer with a pH range of 4–7, and 0.2% DTT). The mixture of proteins labeled with Cy3 and Cy5 was applied to Immobiline DryStrips (pH 4–7, 13 cm) (Cytiva, Tokyo, Japan) and focused on a PROTEAN i12 IEF system (Bio-Rad, Tokyo, Japan). Focused IPG strips were equilibrated and loaded onto 12.5% SDS-polyacrylamide gels (30% acrylamide, 1.5 M Tris-HCl pH 8.8, 10% SDS, 10% ammonium persulfate, and 10% TetraMethylEthyleneDiamine (TEMED)) using low-fluorescence glass plates on an SE 600 Ruby system (GE Healthcare). All electrophoresis procedures were performed in the dark. After SDS-PAGE, gels were scanned using a Pharos FX System (Bio-Rad) with appropriate excitation/emission wavelengths specific for Cy3 (532/605 nm) and Cy5 (635/695 nm). Scanned images were analyzed using the PDQuest Advanced Version 8.0 software (Bio-Rad).
PDQuest was used to identify spots with higher or lower protein expression in ES-2 and KK, and OVMANA and OVTOKO, respectively. Five spots were identified. Mass spectrometric analysis showed that NNMT was present in one of the spots. The spot containing NNMT had higher protein levels in OVMANA and OVTOKO than in KK and ES-2. For further confirmation, ES-2 and OVMANA, ES-2 and OVTOKO, KK and OVMANA, KK, and OVTOKO were labeled with Cy3, Cy5, Cy5, and Cy3, respectively. The protein expression level of the spot containing NNMT was higher in OVMANA than ES-2, OVTOKO than ES-2, OVMANA than KK, and OVTOKO than KK.
Peptide mass fingerprinting (PMF)
All chemicals used in this study were of analytical grade. The compounds 4-Sulfophenyl isothiocyanate, a-cyano-4-hydroxycinnamic acid (CHCA), sodium bicarbonate, and ammonium bicarbonate were purchased from Sigma (St. Louis, MO, USA).
For protein identification by peptide mass fingerprinting, protein spots were excised, digested with trypsin (Promega, Madison, WI), mixed with α-cyano-4-hydroxycinnamic acid in 50% acetonitrile /0.1% TFA, and subjected to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis (Microflex LRF 20, Bruker Daltonics, MA, USA), as described by Fernandez et al. (Electrophoresis 19:1036–1045). Spectra were collected from 300 shots per spectrum over an m/z range of 600–3000 and calibrated by two-point internal calibration using trypsin auto-digestion peaks (m/z 842.5099 and 2211.1046). The peak list was generated using Flex Analysis 3.0. The threshold used for peak-picking was as follows: 500 for the minimum resolution of monoisotopic mass and 5 for S/N. The search program MASCOT, developed by Matrixscience (http://www.matrixscience.com/), was used for protein identification using peptide mass fingerprinting. The following parameters were used for the database search: trypsin as the cleaving enzyme, maximum of one missed cleavage, iodoacetamide (Cys) as a complete modification, oxidation (Met) as a partial modification, monoisotopic masses, and a mass tolerance of ± 0.1 Da. The PMF acceptance criterion included the probability scores.
Western blot analysis
Equal amounts of protein were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membranes were blocked, the primary antibodies were added, and the membranes were incubated with secondary antibodies. Signals were detected using an Image Quant LAS 4000 Mini instrument (GE Healthcare, Wauwatosa, WI, USA).
siRNA
We transfected NNMT siRNA (sc-61213; Santa Cruz Biotechnology) into the OVMANA, RMG1, and ES-2 cell lines cultured in 6-well plates using Lipofectamine RNAi MAX Reagent (13778; Invitrogen, Tokyo, Japan) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific, Tokyo, Japan). Control siRNA-A (sc-37007; Santa Cruz Biotechnology, Tokyo, Japan) was used as a negative control. The knockdown effect was tested by western blotting using an anti-NNMT antibody.
Expression plasmid transfection
We transfected the NNMT Expression Plasmid DNA (RC200641; OriGene Technologies, Inc., MD, USA) into the ES-2 cell line that was cultured in 6-well plates using Lipofectamine 2000 Transfection Reagent (11668019; Thermo Fisher Scientific) and Opti-MEM Reduced Serum Medium (31985070; Thermo Fisher Scientific Tokyo, Japan). The pCMV vector was used as a negative control for all experiments. The cells were incubated for 2 weeks in a medium containing G418 (1000 µg/mL; Thermo Fisher Scientific, Tokyo, Japan). We also established stable control clones expressing NNMT in these cell lines. Drug-resistant clones were further incubated in a medium with puromycin (58-58-2; Sigma-Aldrich, Tokyo, Japan) and tested for the knockdown effect by western blotting using the NNMT antibody.
Statistical analysis
The Student t-test was used to determine the statistical significance of the differences between the comparison groups in vitro. Error bars represent the mean ± standard error of the mean. The relationship between NNMT expression and clinicopathological characteristics was analyzed using Pearson’s χ2 test. Data is equally distributed, and survival rates were calculated using the Kaplan–Meier method and log-rank test.