Roswell Park Memorial Institute-1640 (RPMI-1640) medium, penicillin-streptomycin solution, and trypsin-EDTA (0.05%) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Dulbecco’s modified Eagle’s Media (DMEM; LM001-51) was purchased from Welgene Biotech (Taipei City, Taipei, Taiwan). 6-Gingerol (cat no. 23513-14-6) was purchased from TCI (Tokyo Chemical Industry Co., Tokyo, Japan). Fetal bovine serum (FBS; 12003C) and primary antibodies specific for SOX2 (MAB4423), OCT4 (MABD76), NANOG (MABD24), SF1670 (SML0684), and iron (II) sulfate heptahydrate (F8633) were purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MO, USA). Antibodies specific for β-actin (sc-47778), Wnt5A (sc-365370), BCL-2 (sc-7382), p21 (sc-756), cyclin E (sc-481), and CDK4 (sc-260) and secondary antibodies [anti-mouse (sc-516102) and anti-rabbit (sc-2357)] were obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Next, the Wnt8A (H00007478-B01P) antibody was obtained from Abnova (Taipei City, Taiwan). β-Catenin (#9582), GSK-3β (#9315), BAX (#2772), BCL-xL (#2764), cytochrome C (#11940), p27 Kip1 (#3686), p53 (#9282), pATM (#5883), pATR (#2853), pCHK1 (#2348), pCHK2 (#2197), pBRCA1 (#9009), TCF3/TCF7L1 (#2883), Casp9 (#9502), C-Casp9 (#9505), PTEN (#9188), pAKT (#4060), AKT (#4691), pPI3K (#4228), PI3K (#4257), COX IV (#4850), and GAPDH (#2118) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). TFR1 (ab84036), STEAP3 (ab151566), DMT1 (ab55735), and cyclin D1 (ab6152) antibodies were purchased from Abcam (Cambridge, MA, USA). FPN1 (NBP1-21502) and iNOS (NB300-650) antibodies were obtained from Novus Biologicals (Littleton, CO, USA). Finally, the antibody specific for PD-L1 (R30949) was obtained from NSJ Bioreagents (San Diego, CA, USA).

NCCIT (CRL-2073) and NTERA-2 (CRL-1973) cell lines were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Next, NCCIT cells were cultured and maintained in RPMI-1640 media, and NTERA-2 cells were cultured and maintained in DMEM media plus 10% FBS and 1% penicillin at 37°C in 5% CO2. The medium was changed three times a week after cells reached up to 80% confluence and treated with 6-gingerol. Lastly, the treated cells were incubated at 37°C for 48 h.

Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Then, NCCIT or NTERA-2 cells were maintained in culture media in 96-well culture plates at 3 × 10³ per well (density) for 24 h. Next, cells were incubated in fresh medium containing dimethyl sulfoxide (DMSO) as the vehicle control and treated with 6-gingerol (50–400 μM) for 48 h. Subsequently, MTT (5 mg/mL) was added and incubated for 4 h at 37°C. The resulting formazan product was dissolved in DMSO, and an Ultra Multifunctional Microplate Reader (Tecan, Durham, NC, USA) was used to measure the absorbance at a wavelength of 590 nm. All measurements and experiments were conducted in triplicate.

Protein samples were isolated from untreated (control) or 6-gingerol-treated NCCIT or NTERA-2 cells using radioimmunoprecipitation (RIPA) lysis buffer (20–188; EMD Millipore), which contained protease and phosphatase inhibitors. First, the concentration of proteins was measured using Bradford’s method (Thermo Fisher Scientific). Next, 100 μg of protein from each sample were resolved with sodium dodecyl sulfate-polyacrylamide gel (10%–15%) electrophoresis. Then, the separated proteins were transferred onto nitrocellulose membranes, followed by blocking with 5% skim milk (BD Biosciences, San Jose, CA, USA) in TBS-T buffer [20-μM Tris-HCl (Sigma-Aldrich; Merck KGa A), pH 7.6, 137-μM NaCl (Formedium, Norfolk, UK; NAC03), and 0.1× Tween20 (Scientific Sales, Inc. OakRidge, TN, USA)] for 1 h. Next, the membranes were incubated overnight at 4°C in a shaker with specific primary antibodies diluted in 5% bovine serum albumin (EMDMillipore). Then, the membranes were washed with TBS-T and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Finally, the detection was performed using a Femto Clean Enhanced Chemiluminescence Solution Kit (77449; GenDEPOT, Katy, TX, USA) in a LAS-4000 imaging device (Fujifilm, Tokyo, Japan). Quantifications were conducted using ImageJ software (v.1.8.0_172; National Institutes of Health).

Total RNA was isolated using the RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany) and then quantified using a spectrophotometer at 260 nm. A thermal cycler (C1000 Thermal Cycler; Bio-Rad, Hercules, CA, USA) was used to make cDNA from the total RNA using a first-strand cDNA synthesis kit (Bioneer, Daejeon, Korea) and oligo (dT) primers. PD-L1, p53, and GAPDH cDNA (2-5 µg) were amplified using an RT-PCR Premix Kit (Bioneer) with primers synthesized by Bioneer. The Light Cycler 480II (Roche) was used for qPCR as follows: 2 μL of diluted cDNA was mixed with 10 μL of TB Green Advantage Premix (Takara Bio, Japan) and 1 μL each of the forward and reverse primers. The cycling conditions were as follows: 95°C for 5 min for the initial denaturation, which was followed by 40 cycles of 95°C for 40 s, 58°C for 40 s, 72°C for 40 s, and a final extension of 5 min at 72°C. All reactions were conducted three times and normalized to GAPDH; quantifications were conducted using the obtained Cp values.

The cultured cells were washed with pre-warmed culturing medium supplemented with 10% FBS (staining buffer), and 1 × 10⁶ cells were re-suspended in 1 mL of staining buffer containing MitoTracker Deep Red (40 nM; Invitrogen, Carlsbad, CA, USA: M22426) to measure mitochondrial membrane potential, MitoSOX (5 μM; M36008; Invitrogen) to measure mROS, or CM-H2DCFDA (5 μM; Invitrogen, C6827) to measure cellular ROS. Then, the cells were incubated in a CO2 incubator at 37°C for 30 min, and the stained cells were washed with 1 mL of pre-warmed staining buffer prior to fluorescence-activated cell sorting (FACS). The analysis was performed using FlowJo software.

The DNA content of 6-gingerol-treated and non-treated cells was determined using a BD Cycletest Plus DNA Reagent Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. Approximately 5 × 10⁵ cells, incubated with or without 6-gingerol for 48 h, were washed with PBS and permeabilized with trypsin. RNA interactions with propidium iodide (PI) were neutralized by treating the cells with RNase buffer and trypsin inhibitor. Then, the samples were stained with PI and incubated for 30 min in the dark at room temperature prior to analysis with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA).

The comet assay kit (Abcam, Cambridge, MA, USA) was used to measure cellular DNA damage. This assay is a single-cell gel electrophoresis method for the simple evaluation of cellular DNA damage. First, a base layer of comet agarose was created on a slide, followed by a layer of cells, agarose, and lysis. Next, electrophoresis was performed under neutral conditions, and the cells were stained with DNA dye. Finally, cell morphology was observed by fluorescence microscopy (Olympus IX71/DP72).

Fluorescein-conjugated annexin V (annexin V-FITC) was used to measure apoptosis in NCCIT and NTERA-2 cells. First, the 6-gingerol-treated or untreated cells were washed with PBS and re-suspended in a binding buffer at a concentration of 1 × 10⁶ cells. Then, the cells were stained with annexin V-FITC and PI for 10 min in a dark room at room temperature. Finally, the percentage of apoptotic cells was measured by flow cytometry via FACSCalibur, and the analysis was performed using FlowJo software.

Mitochondria/cytosol fractions from 6-gingerol-treated and non-treated NCCIT and NTERA-2 cells were extracted using a mitochondria/cytosol fractionation kit (Abcam). A 1X cytosol extraction buffer containing DTT and protease inhibitors was added to the cells (5 x 10⁷), which were then incubated on ice for 10 min. After incubation, samples were centrifuged at 700 x g for 10 min at 4°C to collect the supernatant, which was centrifuged again at 10,000 x g for 30 min at 4°C. The supernatant was removed and saved as the cytosol fraction, while the pellet was re-suspended in PBS to obtain the mitochondrial fraction. Next, western blotting of cytochrome c was carried out as described above.

The ATP Determination Kit (Molecular Probes, Eugene, OR, USA) was used to measure ATP. Briefly, NCCIT or NTERA-2 cells were treated with 6-gingerol, and an equal number of cells was collected for the ATP determination assay. The standard reaction solution was made using reaction buffer, DTT, D-luciferin, and firefly luciferase as provided in the kit; then, cells were added along with the standard reaction solution. Luminescence readings were taken immediately using a plate-reading luminometer, and calculations were performed according to the assay protocol.

Iron estimation was performed using an iron assay kit (MAK025) purchased from Sigma-Aldrich (Merck KGaA, St. Louis, MO). Briefly, NCCIT or NTERA-2 cells (2 × 10⁶) treated with or without 6-gingerol were homogenized in an iron assay buffer and collected by centrifugation at 16,000 × g for 10 min at 4°C. Then, the cells were mixed with an iron assay buffer, added to a 96-well plate along with an iron reducer, and incubated in a horizontal shaker for 30 min at 25°C. Then, 100 µL of the iron probe was added to each well and incubated for 1 h at 25°C. Absorbance was measured at 593 nm, and controls were set to 100% for comparison.

After cultured cells (NCCIT or NTERA-2) were washed with culturing medium, 2 mL of staining solution containing FerroFarRed (5 µM; GC903-01; GORYO Chemical) was added prior to incubation in a CO2 incubator at 37°C for 30–40 min. After staining, cells were washed with 1 mL of pre-warmed serum-free culture medium and used for FACS analysis.

All experiments were performed in triplicate. The results were expressed as the mean ± standard error of the mean. Statistical analyses were conducted via the one-way analysis of variance (ANOVA) or Student’s t-test. Additionally, the one-way ANOVA was performed using Tukey’s post hoc test. The analyses were performed using SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA). A p-value < 0.05 (*) was considered statistically significant.

To begin our study, first, we analyzed the proliferation inhibition of 6-gingerol on embryonic CSC viability by the MTT assay. Increasing concentrations of 6-gingerol in NCCIT and NTERA-2 ( Supplementary Figure 1A ) resulted in a concentration-dependent inhibition of cell viability. From these results, we calculated 200 μM 6-gingerol to be the IC₅₀ dosage and selected 100 or 200 μM of 6-gingerol for concentration-dependent studies. We also evaluated the cell morphology of embryonic CSCs after treatment with 6-gingerol using DAPI staining. We observed a decrease in cell number upon 6-gingerol treatment ( Supplementary Figure 1B ). Next, we determined whether 6-gingerol could suppress CSC markers in embryonic CSCs. Evaluation of the CSC markers SOX2, OCT4, and NANOG in embryonic CSCs with 6-gingerol treatment showed a downregulation in the expression patterns of these CSC markers ( Figure 1A ). Then, we confirmed their downregulation in NCCIT and NTERA-2 cells at the mRNA level using real-time qPCR analysis ( Figure 1B ). These results also suggested the ability of 6-gingerol to inhibit CSC proliferation.

Next, we analyzed the expression patterns of Wnt/β-catenin signaling in embryonic CSCs following 6-gingerol treatment. The results showed a downregulation in the expression of Wnt5A, Wnt8A, GSK-3β, β-catenin, and TCF proteins by 6-gingerol treatment in NCCIT and NTERA-2 cells ( Figure 1C ). Then, we confirmed the inhibition of Wnt/β-catenin signaling at the mRNA level ( Figure 1D ). These results suggested the capability of 6-gingerol against embryonic CSCs. We also analyzed the ability of 6-gingerol to inhibit tumor invasion in embryonic CSC using the Matrigel invasion assay. Results indicated a successful inhibition of tumor invasion in NCCIT and NTERA-2 cells by 6-gingerol treatment ( Supplementary Figure 1C ). These results suggested the overall cytotoxic activity of 6-gingerol against embryonic cancer stem cells.

We hypothesized that the anti-tumor activity of 6-gingerol might begin by generating ROS. To analyze this, we measured iNOS expression following 6-gingerol treatment. Our results showed that increasing concentrations of 6-gingerol upregulated iNOS expression in NCCIT and NTERA-2 cells ( Figure 2A ). We confirmed this by measuring the expression of iNOS mRNA level in embryonic CSCs after treatment with 6-gingerol with similar results ( Figure 2B ). The induction of iNOS suggested that 6-gingerol may cause ROS generation in embryonic CSCs. As we expected, we found that 6-gingerol treatment successfully induced cellular ( Figure 2C ) and mitochondrial ( Figure 2D ) ROS, which suggested ROS as the reason for the anti-tumor activity of 6-gingerol.

Then, we verified the ability of 6-gingerol to induce the DNA damage response (DDR) in NCCIT and NTERA-2 cells. To analyze this, we used a comet assay to determine DNA double-strand breaks, which showed that 6-gingerol induced DNA double-strand breaks in NCCIT and NTERA-2 cells ( Supplementary Figure 2A ). Moreover, we observed a significant increase in the comet length and number of comet-positive cells in 6-gingerol-treated cells compared with non-treated control cells ( Supplementary Figure 2B ). These results hinted at the DDR induction of 6-gingerol in embryonic CSCs. To confirm this, we evaluated the expression levels of proteins involved in the DDR and found an increase in the expression of phosphorylated histone, ATM, ATR, CHK1, CHK2, and BRCA1 following 6-gingerol treatment in NCCIT and NTERA-2 cells ( Supplementary Figure 2C ). These results suggested that either ATM or ATR may act as a key regulator of DDR induction by 6-gingerol.

Based on previous results, we showed that 6-gingerol induces ROS and the DDR in embryonic CSCs. Therefore, we analyzed the impact of 6-gingerol on the cell cycle and apoptosis induction. Flow cytometry of embryonic CSCs treated with 6-gingerol showed an arrest in the G0/G1 phase of the cell cycle ( Supplementary Figure 3A ). These results indicated that DDR induction leads to prolonged cell cycle arrest, and to confirm this, we analyzed cell cycle checkpoint protein levels by Western blot. These results showed an elevation in the expression of tumor suppressor proteins p21 and p27 as well as a decrease in the expression of cyclin D1, cyclin E, and CDK4 proteins ( Supplementary Figure 3B ). Then, we confirmed these results by measuring the transcriptional expression of CCND1, CCNE1, CDK4, CDKN1A, and CDKN1B genes ( Supplementary Figure 3C ), which confirmed the induction of a cell cycle arrest. These results indicated that 6-gingerol causes cell cycle arrest in embryonic CSCs and may also induce apoptosis.

Next, we evaluated apoptosis induction by 6-gingerol in embryonic CSCs using flow cytometry. The results showed that 6-gingerol induced apoptosis in NCCIT and NTERA-2 cells ( Figure 3A ). Then, we investigated the apoptosis pathway by measuring protein levels of the key apoptosis regulators BCL2 associated X (BAX), B-cell lymphoma 2 (BCL-2), B-cell lymphoma-extra-large (BCL-xL), cleaved caspase 9, and cytochrome c ( Figure 3B ). These results showed a downregulation in the expression levels of BCL-2 and BCL-xL while BAX, cleaved caspase 9, and cytochrome c were upregulated upon 6-gingerol treatment. These data suggested a possible induction of the intrinsic apoptosis pathway. As intrinsic apoptosis depends heavily on the BAX/BCL-2 ratio, we confirmed the expression of BAX, BCL-2, and CASPASE 9 mRNA following 6-gingerol treatment in embryonic CSCs ( Figure 3C ). These results suggested a possible release of cytochrome c from the mitochondria into the cytosol, which was confirmed by comparing cytochrome c levels in the cytosol and mitochondria ( Figure 3D ). These results showed a decrease in the amount of cytochrome c in mitochondria and a corresponding increase in the cytosol. Also, we analyzed the ATP concentration of embryonic CSCs after treatment with 6-gingerol ( Figure 3E ); the results indicated a decrease in ATP production, which corresponds with a 6-gingerol induction of the intrinsic apoptosis pathway.

Taken together, these data indicate the induction of intrinsic apoptosis by 6-gingerol in embryonic CSCs. To identify the molecular signaling responsible for this mechanism, we analyzed the expression of tumor suppressor protein PTEN and its downstream targets PI3K, AKT, and p53. We found elevated expression of PTEN and p53 while phosphorylated PI3K and AKT were downregulated by 6-gingerol treatment of NCCIT and NTERA-2 cells ( Figure 4A ). Also, we analyzed the expression of PD-L1, as PI3K/AKT/p53 signaling may regulate its expression. Our results suggested the same, as 6-gingerol suppressed the expression levels of PD-L1 in embryonic CSCs. This result hinted at the role of PTEN/PI3K/AKT/p53 signaling in PD-L1 inhibition by 6-gingerol. To confirm this signaling and its effect on PD-L1 expression, we repeated our experiment with a specific inhibitor of PTEN (SF1670). We found increased phospho-PI3K/AKT and PD-L1 expression upon SF1670 treatment, and 6-gingerol successfully inhibited the expression of these proteins ( Figure 4B ). The expression of p53 was suppressed with PTEN inhibitor treatment and rescued by the addition of 6-gingerol in embryonic CSCs, which suggested the ability of 6-gingerol to induce PTEN and p53 expression and thereby inhibit PD-L1 expression. It also suggested a role for PTEN in the regulation of PD-L1 during 6-gingerol treatment.

We found that 6-gingerol can induce intrinsic apoptosis in embryonic CSCs. Next, we analyzed the mechanism of these activities and hypothesized that iron homeostasis might play a role. First, we estimated the total iron concentration in NCCIT and NTERA-2 cells and media with or without 6-gingerol treatment using an iron assay kit ( Figure 5A ). The results showed an increase in the total iron concentration in the medium following 6-gingerol treatment. However, the concentration of iron in 6-gingerol-treated cells was significantly reduced. This suggested an enhanced iron release into the medium. We confirmed this by using flow cytometry to estimate the ferrous ion (Fe2+) concentration in NCCIT and NTERA-2 cells after treatment with 6-gingerol; results suggested a significant decrease in the Fe2+ ion concentration following 6-gingerol treatment ( Figure 5B ). These results indicated an upregulation in iron transport by the conversion of Fe2+ to Fe3+, which highlighted the conversion of total iron for iron metabolism.

To confirm this, we analyzed the expression levels of proteins responsible for iron transport, and the results showed downregulated expression of TFR1 and ferroportin (FPN1), which transports iron ( Figure 5C ). Also, we found that 6-gingerol suppressed the expression of DMT1 and STEAP3, helping iron conversion. These results suggested a role of iron metabolism in the anti-cancer activity of 6-gingerol against embryonic CSCs. Next, we analyzed the role of iron metabolism in PTEN induction and p53/PD-L1 signaling. For this, we added iron sulfate (FeSO₄) to NCCIT and NTERA-2 cells with or without 6-gingerol treatment. Supplementation with FeSO₄ resulted in suppressed PTEN and p53 expression, which was accompanied by an elevation in PD-L1 expression ( Figure 5D ). The addition of 6-gingerol reversed these expression patterns, which suggested a role for iron metabolism in PTEN-mediated p53 and PD-L1 expressions following 6-gingerol treatment in embryonic CSCs.

We found that iron metabolism and PTEN/p53/PD-L1 signaling may play a role in the induction of apoptosis by 6-gingerol in embryonic CSCs. Here, we analyzed the potential role of microRNA in the apoptotic activity of 6-gingerol and the role of iron metabolism in microRNA expression in the presence of 6-gingerol. First, we measured the expression levels of miR-20b, miR-21, and miR-130b, which play key roles in PTEN-mediated PD-L1 expression. The mRNA analysis showed a downregulation of miR-20b, miR-21, and miR-130b following 6-gingerol treatment in NCCIT and NTERA-2 cells ( Figure 6A ). These data indicated a potential role for these microRNAs in apoptosis induction by 6-gingerol. To confirm this activity, we used the PTEN inhibitor SF1670 and analyzed the expression of these microRNAs in the presence of 6-gingerol ( Figure 6B ). The results showed elevated expression of miR-20b, miR-21, and miR-130b with PTEN inhibitor treatment, which was downregulated by 6-gingerol treatment. These results suggested a role for PTEN in regulating these microRNAs.

Next, we verified the role of iron metabolism in the expression of miR-20b, miR-21, and miR-130b by supplementing FeSO₄ with or without 6-gingerol. These results also showed an increase in the expression of miR-20b, miR-21, and miR-130b following FeSO₄ treatment, which were reversed by the addition of 6-gingerol in both NCCIT and NTERA-2 cells ( Figure 6C ). These results contributed to evidence that iron metabolism plays a role in the induction of cell death by regulating microRNA expression following 6-gingerol treatment.