Cell culture and drug-tolerant persister cancer cell derivation
HNC cell lines, namely AMC HN3 and HN4, were used for our experiments [16]. The cell lines were authenticated by short tandem repeat-based DNA fingerprinting and multiplex polymerase chain reaction (PCR). The cells were cultured in Eagle's minimum essential medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Drug-tolerant persister cancer cells were derived from HN3 and HN4 cells with 10 nM paclitaxel (Sigma-Aldrich, St. Louis, MO, USA). Paclitaxel was treated for 6 days with a new drug added every 3 days and this was repeated in regrown cells after no treatment for 38 days. Re-derived persister and parental cells were used in experiments. PCC was selected by 10 nM paclitaxel every 2 weeks to maintain drug tolerance characteristics.
Cell viability and death assays
Cell viability was measured in the cells that were subjected to (1S,3R)-RSL3) (19288; Cayman Chemical Co., Ann Arbor, MI, USA), erastin (S7242; Selleckchem, Houston, TX, USA), sulfasalazine (S0883; Sigma-Aldrich), or an equivalent amount of dimethyl sulfoxide (DMSO), or were cultured in the conditioned media with no cysteine and cystine (cyst(e)ine, 1641454; MP Biomedicals, Irvine, CA, USA). After exposure, cell viability was assessed using cell counting kit-8 (CCK-8) (CK04; Dojindo Molecular Technologies, Inc., Tokyo, Japan) according to the manufacturer's protocol. The cells were incubated with the CCK-8 solution for 1 h, and then the cell viability was measured at the absorbance of 450 nm using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA, USA).
After exposure to the agents, cell death was assessed via SYTOX Green (S34860; Thermo Fisher Scientific) staining. The samples were washed three times with Hanks' balanced salt solution without calcium and magnesium (HBSS, 14025076; Thermo Fisher Scientific), followed by staining of cells in each plate with 5 µM SYTOX Green in HBSS for 20 min. The stained cells were observed using a ZEISS fluorescent microscope (Axiovert 200M; Oberkochen, Germany) and analyzed using ImageJ software (NIH, Bethesda, MD, USA). The mean SYTOX Green-positive fractions were compared with those of the control group.
Measurement of lipid and mitochondrial reactive oxygen species
Lipid reactive oxygen species (ROS) generation was measured by adding 5 µM BODIPY™ 581/591 C11 (a lipid peroxidation sensor, D3861; Thermo Fisher Scientific) for 30 min at 37°C. The ROS levels were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA), (non)oxidized and oxidized forms were confirmed by ZEISS fluorescent microscope. Image quantification was performed using ImageJ software. For mitochondrial ROS, cells were seeded in 60 mm dishes. After indicated drug treatment, cells were incubated with 5 µM mitoSOX™ Red (M36008; Thermo Fisher Scientific) for 20 min. MitoSOX™ Red was measured by ZEISS fluorescent microscope. The quantification of fluorescence intensity was performed using ImageJ software.
Glycolysis and glutamate assays
Glycolysis assay was measured using a glycolysis assay kit (ab197244; Abcam) at 380 nm excitation and 615 nm emission using a SpectraMax M2 microplate reader. The glycolytic effect was calculated through extracellular acidification (ECAR) using a microplate fluorometer at 15 min intervals and was examined from ECAR assay at 120 min. All examinations were operated on in 5×105 cells per sample following the manufacturer’s protocol. Glutamate contents were measured using a glutamate assay kit (ab83389; Abcam) following the manufacture’s protocol.
Measurement of GSH synthesis and intracellular iron
Intracellular GSH levels in HNC cell lysates were measured using a GSH/GSSG assay kit (EGTT-100; BioAssay Systems, Hayward, CA, USA) according to the manufacturer's instructions.
Labile iron pool (LIP) assay was measured by using calcein acetoxymethyl ester (354217; Corning Inc., Corning, NY, USA) and iron chelator, deferoxamine (ab120727; Abcam, Cambridge, UK). The cells were loaded with calcein-AM (8 µg/ml) for 30 min at 37°C and then washed with HBSS. Deferoxamine was added at a final concentration of 100 µM to remove iron from calcein, causing dequenching. The change in fluorescence following the addition of deferoxamine was used as an indirect measure of the LIP. Fluorescence was measured at 485 nm excitation and 535 nm emission with a VICTOR X3 microplate reader (PerkinElmer, Waltham, MA, USA) and ZEISS fluorescent microscope.
Measurements of autophagic flux
The cells were seeded and were treated with erastin or other agents with or without 30 nM Wortmannin (W1628; Sigma-Aldrich). Also, autophagy-related molecules were confirmed by immunoblotting. To assess the later process of autophagy, all cells were stained with Lysotracker™ Red DND-99 (L7528; Thermo Fisher Scientific). Co-localization of LC3-GFP-puncta and lysosome was confirmed using the ZEISS LSM 880 confocal microscope.
Measurements of free fatty acid
For quantification of free fatty acid (FFA), parental HNC cells and PCC were seeded in 100 mm dishes. Then, cells were treated with or without ferroptosis inducers. Intracellular FFA was measured using PicoSens™ Free Fatty Acid Quantification Kit (BM-FFA-100; BIOMAX, Seoul, Republic of Korea) according to the manufacturer's instructions.
GC/MS analysis
PCC and parental cells were seeded in culture medium in 150-cm tissue culture dishes and were harvested in two days with a rubber-tipped cell scraper. The cells were washed, centrifuged twice with 1× phosphate-buffered saline and transferred to tubes in an equal number of 5×106 cells. The cells proceeded with lipid extraction after flash-freezing in LN2. For fatty acid methyl esters (FAME) analysis, cells were lyophilized and ground into fine powders. Fatty acids were extracted using 2 mL methylation mixture (MeOH:Benzene:DMP (2,2-Dimethoxy-propane):H2SO4 = 39:20:5:2) and 1 mL heptane, 80 ℃ for 2 h. Then, supernatants were analyzed using GC/MS (Agilent 7890 GC System; Agilent Technologies, Santa Clara, CA, USA). The analysis condition was as follows: column (DB-23, 120 mm*0.25 mm* 0.25 µm; Agilent), injector (250 ℃), detector (FID-280 ℃, H2 35 ml/min, air 350 ml/min, He 10 ml/min), STD (CRM47885; Supelco 37 component FAME Mix; Supelco, Inc., Bellefonte, PA, USA), and ISTD (P6125; Pentadecanoic acid) (Sigma-Aldrich) [17]. After GC/MS analysis, all samples were normalized.
RNA interference and gene transfection
HNC cells were seeded for gene silencing or overexpression. Cells were transfected 24 h later with 10 nmol/L small-interfering RNA (siRNA) targeting human SIRT1 or scrambled control siRNA (Integrated DNA Technologies, Coralville, IA, USA) using Lipofectamine RNAiMAX reagent (13778075; Thermo Fisher Scientific). PCC was stably transduced with short hairpin RNA (shRNA) targeting PGRMC1 (pGPU6/Neo, GenePharma, Shanghai, China) using Lipofectamine 3000 reagent (L3000001; Thermo Fisher Scientific). HN3 and HN4 cells were seeded and stably transfected with a control pcDNA3.1 plasmid (V790-20; Addgene, Watertown, MA, USA) or pcDNA3.1-PGRMC1 plasmid by using Lipofectamine 3000 reagent. The levels of PGRMC1 and SIRT1 expression were confirmed via western blotting. pEGFP-LC3 (21073; Addgene) was stably transfected into HN4 parental cells, HN4-PGRMC1 plasmid, HN4PCC, and HN4PCC-shPGRMC1 using Lipofectamine 3000 reagent.
Reverse transcription-quantitative PCR and methylation-specific PCR
HNC cells were cultured with 70% confluence in 6-cm tissue culture dishes. Total RNA from HNC cells was isolated using a total RNA extraction kit (K-3140; Bioneer, Daejeon, Republic of Korea) following the manufacturer's instructions. A reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a SensiFAST™ SYBR® No-ROX Kit (BIO-98050; Bioline International, Toronto, Canada) after cDNA synthesis with a SensiFAST™ cDNA Synthesis Kit (BIO-65054; Bioline International). PGRMC1, SIRT1, and ACTB were amplified, and the relative target mRNA levels were determined using mathematical expression 2−(ΔΔCt). All data were normalized against ACTB mRNA levels. Real-time PCR was performed with ViiA™ 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Methylation-specific PCR (MSP) indicated methylated promoter level in bisulfite-treated genomic DNA. Genomic DNA from HNC cells was extracted by a genomic DNA extraction kit (YGB100; Real Biotech Co., Taipei, Taiwan). Extracted genomic DNA was converted into a bisulfite form using a BisulFlash DNA Modification Kit (P-1026-050; EpiGentek, Farmingdale, NY, USA). The degree of methylation was determined in SIRT1 by RT-qPCR using a Methylamp MS-qPCR Fast Kit (P-1028-100; EpiGentek).
Immunoblotting and immunostaining
Cells were plated and grown with 70% confluence and then treated with indicated drugs or not. Cells were lysed at 4°C in a cell lysis buffer (9803; Cell Signaling Technology, Danvers, MA, USA) with a protease/phosphatase inhibitor cocktail (5872; Cell Signaling Technology). A total of 10–40 µg protein was resolved by SDS-PAGE on 10–15% gels; the resolved proteins were then transferred to nitrocellulose or polyvinylidene difluoride membranes and probed with primary and secondary antibodies. The following primary antibodies were used: PGRMC1 (K004086P; Solarbio Life Science, Beijing, China), CD36 (K004214P; Solarbio), ATGL (K004384P; Solarbio), PLIN2 (K004402P; Solarbio), ACC (3662; Cell Signaling Technology Co., Danvers, MA, USA), FASN (K001685P; Solarbio), CPT1A (K000391P; Solarbio), AMPK (2532; Cell Signaling), pAMPK (2531; Cell Signaling), 4-HNE (MA5-27570; Invitrogen), LC3B (K002189P; Solarbio), ATG5 (K106671P; Solarbio), p62 (K005444P; Solarbio), SIRT1 (sc74465; Santa Cruz Biotechnology, Santa Cruz, CA, USA), TTL (K009740P; Solarbio), TCP1 (K003097P; Solarbio), tyrosinated α-tubulin (ABT171; Merck Millipore, Burlington, MA, USA), detyrosinated α-tubulin (AB3201; Merck Millipore), xCT (K009230P; Solarbio), GPX4 (K006597P; Solarbio), ACSL4 (K004812P; Solarbio), and β-actin (BS6007M; BioWorld, Atlanta, GA, USA). β-actin served as the total loading control.
The cells were also immunostained with an antibody against PGRMC1 (1:200; Solarbio) or detyrosinated α-tubulin (1:200; Merck Millipore). The cells were co-stained with BODIPY™ 493/503 for lipid droplets (D3922; Thermo Fisher Scientific), LysoTracker™ Deep Red (L12492; Thermo Fisher Scientific), or MitoTracker TM (M7510; Thermo Fisher Scientific). Nuclei were blue-stained with 4',6-diamidino-2-phenylindole (DAPI).
Tumor xenograft
All animal study procedures were performed by protocols approved by the Institutional Animal Care and Use Committee (IACUC). Six-week-old athymic BALB/c male nude mice (nu/nu) were purchased from OrientBio (Seoul, Republic of Korea). HN4 cells with transfection of PGRMC1 overexpression or control vector and HN4PCC with shPGRMC1 or control vector were subcutaneously injected into the bilateral flank of nude mice. From the day when gross nodules were detected in tumor implants, mice were subjected to different treatments: vehicle or sulfasalazine (250 mg/kg daily per intraperitoneal route) [18]. Erastin was not used due to its physiological instability and instead, sulfasalazine was used in our in vivo experiment [4]. Each group included six mice. The tumor size and weight of each mouse were measured twice a week, and tumor volume was calculated as (length × width2)/2. After mice were sacrificed, tumors were isolated and analyzed by staining lipid droplets. The values were compared among differently treated tumors.
The Cancer Genome Atlas (TCGA) dataset and statistical analysis
The expression levels of PGRMC1 mRNA were obtained from the normal mucosa (n = 44) and HNC (n = 499) datasets of TCGA. The tumor and survival data were analyzed to find the correlation between the expression level of PGRMC1 mRNA and their survival outcomes.
Data were presented as mean±standard deviation (s.d.). The statistically significant differences between the treatment groups were assessed using Mann–Whitney U-test or analysis of variance (ANOVA) with the Bonferroni post-hoc test. The median values of low and high expression levels of PCBP1 mRNA were determined and compared using a t-test. The cutoff value of PGRMC1 was determined at the lowest P values for overall survival. Univariate Cox proportional hazards regression analyses were used to identify associations between PGRMC1 mRNA expression levels and overall survival in the HNC cohort. The Kaplan–Meier and log-rank tests were used to determine and statistically compare the survival rates, respectively. All statistical tests were two-sided, and a P-value of < 0.05 was considered to be statistically significant. The statistical tests were performed using IBM SPSS Statistics version 22.0 (IBM, Armonk, NY, USA).