The human breast cancer cell lines (MDA-MB-453 and MCF-7) were purchased from the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (HyClone, USA) containing 10% fetal bovine serum (FBS; Capricorn, Germany) and 2 mM L-glutamine at 37oC in an incubator with a humidified atmosphere containing 5% CO2.
Both MDA-MB-453 and MCF-7 cells were treated with curcumin (purity>98%; Sigma-Aldrich, USA) dose 0, 1, 2, 5, 10, 20, and 50 μΜ for 48 h or with 10 μM Erastin as a control for 24 h. After incubation, both cell viability was examined using cell counting kit-8 (CCK-8).
A total of 2×105 cells were seeded per well and grown to 40-60% confluence. The SLC1A5 siRNA (si-SLC1A5), and blank plasmid (si-NC) were purchased from GenePharma (Shanghai, China). Vectors were transfected into both MDA-MB-453 and MCF-7 cells using LipofectamineÒ 3000 kit (Invitrogen, USA) according to the manufacturer’s protocols. After 48 h transfection, the transfection efficiency was determined by western blot.
Cell viability analysis
100 μL of cell suspension (5×103 cells) were plated in 96-well plates for incubation at 37 °C with 5 % CO2. When 70% of confluence was reached, cells were treated with curcumin or transfected with si-SLC1A5. Twenty-four hours after transfection, 10 μL CCK-8 solution (Beyotime Biotechnology, Nantong, China) was added to each well and incubated for 60 min. Then the absorbance of each well was assessed at 450 nm using a microplate reader (Thermo Fisher, Massachusetts, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted with TRIzol reagent (Invitrogen, USA) according to the supplier’s instructions. We reverse-transcribed 1 μg of total RNA to single-strand complementary DNA (cDNA) using the One Step PrimeScript miRNA cDNA Synthesis Kit (TaKaRa, Dalian, China). qRT-PCR was performed in triplicate using SYBR Green PCR Master Mix (Life Technologies, USA) following the manufacturer's protocols. The relative expression of acyl-CoA synthetase long-chain family member 4 (ACSL4), nicotinamide adenine dinucleotide phosphate-oxidase 1 (NOX1), glutathione peroxidase 4 (GPX4), and Ferritin was normalized to β-actin, and values were calculated using the 2-ΔΔCt method. The sequences of primers for qPCR were: ACSL4 forward:5’-TTTTGCGAGCTTTCCGAGTG-3’, reverse: 5’-AGCCGACAATAAAGTACGCAA-3, NOX1 forward: 5’-TTGGGTCAACATTGGCCTGT-3’, reverse:5’-AAGGACAGCAGATTGCGACA-3’, GPX4 forward:5’-ATTGGTCGGCTGGACGAG-3’, reverse: 5’-TCGATGTCCTTGGCGGAAAA-3’, FTC forward 5’-GCCACTTCTTCCGCGAATTG-3’, reverse: 5’-TTCATGGCGTCTGGGGTTTT-3’, β-actin forward: 5’-TCCCTGGAGAAGAGCTACGA-3’, reverse: 5’-AGCACTGTGTTGGCGTACAG-3’.
To extract total protein, cells were lysed in a RIPA Lysis Buffer (Beyotime Biotechnology, Nantong, China). The lysate was centrifuged at 12,000 rpm at 4 ℃ for 10 min. The supernatant was transferred to a new tube to quantify the protein amount using the BCA assay. Subsequently, electrophoresis was conducted with 12% SDS-PAGE followed by transfer onto polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% (w/v) non-fat dry milk, the membranes were incubated with primary antibodies against β-actin (1:1000 dilution), SLC1A5 (1:1000 dilution), GOT1 (1:1000 dilution), and GLS2 (1:1000 dilution). All antibodies were purchased from Abcam (Shanghai, China). Then, the appropriate HRP-conjugated secondary antibodies (1:5000 dilution, Proteintech, Wuhan, China) were applied. The protein bands were detected using a chemiluminescence procedure (Pierce, Rockford, IL, USA) on a Tanon 5200 Imaging system (Shanghai, China). Densitometric analysis was performed using Image-Pro Plus 6.0 software (Media Cybernetics, USA). The expression levels of protein in each sample were normalized to β-actin.
Nude mice model
Female BALB/c nude mice at 6-8 weeks of age were purchased from Guangdong Medical Laboratory Animal Center (Foshan, China), and animal model experiments were approved by the Ethical Committee of The Affiliated Hospital of Qingdao University. MCF-7 cells (5×106) were suspended in serum-free DMEM medium and then injected into the right posterior flanks of mice. After two weeks, a total of 20 mice were randomly divided into two groups (n=10 per group). Mice in the curcumin administration group were treated with 30 mg/kg/d curcumin (Intragastric administration). Mice in the control group were fed with 0.9% sodium chloride plus 1% DMSO. The tumor growth in the mice was examined every three days. Mice were sacrificed by intraperitoneal injection of pentobarbital sodium (200 mg/kg) after four weeks of curcumin administration, and the size of each tumor was measured, and tumor tissues were collected for further experiments. The expression of SLC1A5 (1:500 dilution, Cell Signaling Technology, USA) and Ki-67 (1:500 dilution, Cell Signaling Technology, USA) was evaluated by immunohistochemistry staining according to the manufacturer’s instructions and previous studies .
Intracellular ferrous iron (Fe2+) level was determined using the iron assay kit purchased from Abcam (#ab83366) and was used according to the manufacturer’s instructions. The experiment for each group was repeated three times.
Malondialdehyde (MDA) assay
The intracellular MDA concentration in cell lysates or tissues was assessed using a Lipid Peroxidation Assay Kit (#ab118970, Abcam) according to the manufacturer’s instructions. Briefly, the MDA in the sample reacted with thiobarbituric acid (TBA) to generate a MDA-TBA adduct. The MDA-TBA adduct was quantified colorimetrically (optical density (OD) = 532 nm). The experiment for each group was repeated three times.
Glutamine uptake assay
Breast cancer cells were cultured in six-well plates in glutamine-free DMEM/F-12 medium (Invitrogen, USA). After collecting and counting, cells were incubated with 200 nM [3H]-L-glutamine (PerkinElmer, USA) in glutamine-free DMEM/F-12 medium for 15 min at 37 °C in the presence of curcumin or curcumin+SLC1A5 siRNA. Cells were collected, transferred to filter paper using a 96-well plate harvester (Wallac PerkinElmer), dried, exposed to scintillation fluid, and counts measured using a liquid scintillation counter (PerkinElmer).
Determination of lipid ROS levels
Cells (2×105) were seeded in a 6-well plate and were treated with curcumin or Erastin for 24 h according to previous studies, and all cells were cultured in DMEM medium with 5 μM BODIPY-C11 (Thermo Fisher Scientific, USA) for 45 min at room temperature. After that, cells were collected and washed twice with PBS buffer. Subsequently, cells were re-suspended in 500 μL PBS, and then filtered on a 0.4 μm nylon cell strainer and subjected to the flow cytometry analysis to detect the amount of ROS within cells. The fluorescence intensities of cells were determined using a FACSCalibur flow cytometer (BD Bioscience, USA). Each experiment was repeated three times.
All experiments were repeated at least three times. Data are presented as mean ± SD. Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software, Inc., USA). Unpaired Student's t-tests were used to compare the means of two groups and one-way ANOVA was used to compare three or more groups. Pairwise group comparisons were conducted using Tukey's test as a post-hoc test following ANOVA. P<0.05 was considered statistically significant.